Factor VII or VIIa-like molecules

ABSTRACT

Conjugates of Factor VII (FVII) and Factor VIIa (FVIIA) are provided, as are methods for preparing them. Methods for producing novel polypeptides contributing to the production of such conjugates are provided. Methods of treatment by administering a FVII or FVIIa conjugate are provided.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application claims priority to and benefit of the followingUnited States and international patent applications: Danish PatentApplication Number PA 2000 00218, filed Feb. 11, 2000; U.S. ProvisionalPatent Application No. 60/184,036, filed Feb. 22, 2000; and, U.S.Provisional Patent Application No. 60/241,916, filed Oct. 18, 2000.

COPYRIGHT NOTIFICATION

Pursuant to 37 C.F.R. 1.71(e), Applicants note that a portion of thisdisclosure contains material which is subject to copyright protection.The copyright owner has no objection to the facsimile reproduction byanyone of the patent document or patent disclosure, as it appears in thePatent and Trademark Office patent file or records, but otherwisereserves all copyright rights whatsoever.

FIELD OF THE INVENTION

The present invention relates to novel Factor VII (FVII) or Factor VIIa(FVIIa) polypeptide conjugates, to their preparation and use in therapy,in particular for the treatment of a variety of coagulation-relateddisorders.

BACKGROUND OF THE INVENTION

Blood coagulation is a process consisting of a complex interaction ofvarious blood components (or factors) that eventually gives rise to afibrin clot. Generally, the blood components participating in what hasbeen referred to as the coagulation “cascade” are proenzymes orzymogens, i.e., enzymatically inactive proteins that are converted intoan active form by the action of an activator. One of these coagulationfactors is FVII.

FVII is a vitamin K-dependent plasma protein synthesized in the liverand secreted into the blood as a single-chain glycoprotein with amolecular weight of 53 kDa (Broze & Majerus, J. Biol. Chem 1980;255:1242-1247). The FVII zymogen is converted into an activated form(FVIIa) by proteolytic cleavage at a single site, R152-I153, resultingin two chains linked by a single disulfide bridge. FVIIa in complex withtissue factor (FVIIa complex) is able to convert both factor IX andfactor X into their activated forms, followed by reactions leading torapid thrombin production and fibrin formation (Osterud & Rapaport, ProcNatl Acad Sci USA 1977; 74:5260-5264).

FVII undergoes post-translational modifications, including vitaminK-dependent carboxylation resulting in ten γ-carboxyglutamic acidresidues in the N-terminal region of the molecule. Thus, residue number6, 7, 14, 16, 19, 20, 25, 26, 29 and 35 shown in SEQ ID NO:1 areγ-carboxyglutamic acids residues in the Gla domain important for FVIIactivity. Other post-translational modifications include sugar moietyattachment at two naturally occurring N-glycosylation sites at position145 and 322, respectively, and at two naturally occurringO-glycosylation sites at position 52 and 60, respectively.

The gene coding for human FVII (hFVII) has been mapped to chromosome 13at q34-qter 9 (de Grouchy et al., Hum Genet 1984; 66:230-233). Itcontains nine exons and spans 12.8 Kb (O'Hara et al., Proc Natl Acad SciUSA 1987; 84:5158-5162). The gene organisation and protein structure ofFVII are similar to those of other vitamin K-dependent procoagulantproteins, with exons 1a and 1b encoding for signal sequence; exon 2 thepropeptide and Gla domain; exon 3 a short hydrophobic region; exons 4and 5 the epidermal growth factor-like domains; and exon 6 through 8 theserine protease catalytic domain (Yoshitake et al., Biochemistry 1985;24: 3736-3750).

Reports exist on experimental three-dimensional structures of hFVIIa(Pike et al., PNAS. U.S.A., 1999; 96:8925-30 and Kemball-Cook et al., J.Struct. Biol, 1999; 127:213-223), of hFVIIa in complex with solubletissue factor using X-ray crystallographic methods (Banner et al.,Nature, 1996; 380:41 and Zhang et al., J. Mol. Biol, 1999; 285: 2089),and of smaller fragments of hFVII (Muranyi et al., Biochemistry, 1998;37:10605 and Kao et al., Biochemistry, 1999; 38:7097).

Relatively few protein-engineered variants of FVII have been reported(Dickinson & Ruf, J Bio Chem, 1997; 272:19875-19879, Kemball-Cook etal., J Biol Chem, 1998; 273:8516-8521, Bharadwaj et al., J Biol Chem,1996; 271:30685-30691, Ruf et al., Biochemistry, 1999; 38:1957-1966).

Reports exist on expression of FVII in BHK or other mammalian cells(WO92/15686, WO91/11514 and WO88/10295) and co-expression of FVII andkex2 endoprotease in eukaryotic cells (WO 00/28065).

Commercial preparations of human recombinant FVIIa are sold asNovoSeven®. NovoSeven® is indicated for the treatment of bleedingepisodes in hemophilia A or B patients. NovoSeven® is the only rFVIIafor effective and reliable treatment of bleeding episodes available onthe market.

An inactive form of FVII in which arginine 152 and/or isoleucine 153is/are modified has been reported in WO91/1154. These amino acids arelocated at the activation site. WO 96/12800 describes inactivation ofFVIIa by a serine proteinase inhibitor; inactivation by carbamylation ofFVIIa at the α-amino acid group I153 has been described by Petersen etal., Eur J Biochem, 1999; 261:124-129. The inactivated form is capableof competing with wild-type FVII or FVIIa for binding to tissue factorand inhibiting clotting activity. The inactivated form of FVIIa issuggested to be used for treatment of patients being in hypercoagulablestates, such as patients with sepsis, in risk of myocardial infarctionor of thrombotic stroke.

A circulating rFVIIa half-life of 2.3 hours was reported in “SummaryBasis for Approval for NovoSeven®,” FDA reference number 96-0597.Relatively high doses and frequent administration are necessary to reachand sustain the desired therapeutic or prophylactic effect. As aconsequence adequate dose regulation is difficult to obtain and the needof frequent intravenous administrations imposes restrictions on thepatient's way of living.

Another problem in current rFVIIa treatment is the relative instabilityof the molecule with respect to proteolytic degradation. Proteolyticdegradation is a major obstacle for obtaining a preparation in solutionas opposed to a lyophilised product. The advantage of obtaining a stablesoluble preparation lies in easier handling for the patient, and, in thecase of emergencies, quicker action, which potentially ran become lifesaving. Attempts to prevent proteolytic degradation by site directedmutagenesis at major proteolytic sites have been disclosed inWO88/10295.

A molecule with a longer circulation half-life would decrease the numberof necessary administrations. Given the association of current FVIIaproduct with frequent injections, and the potential for obtaining moreoptimal therapeutic FVIIa levels with concomitant enhanced therapeuticeffect, there is a clear need for improved FVII or FVIIa-like molecules.

One way to increase the circulation half-life of a protein is to ensurethat renal clearance of the protein is reduced. This can be achieved byconjugating the protein to a chemical moiety, which is capable ofconferring reduced renal clearance to the protein.

Furthermore, attachment of a chemical moiety to the protein orsubstitution of amino acids exposed to proteolysis can effectively blocka proteolytic enzyme from contact leading to proteolytic degradation ofthe protein. Polyethylene glycol (PEG) is one such chemical moiety thathas been used in the preparation of therapeutic protein products.

WO98/32466 suggests that FVII, among many other proteins, can bePEGylated but does not provide any further information in this respect.

SUMMARY OF THE INVENTION

This application discloses improved FVII and FVIIa molecules, inparticular recombinant hFVII and hFVIIa molecules, providing one or moreof the aforementioned desired benefits. Thus, the conjugate of thepresent invention has one or more improved properties as compared tocommercially available rFVIIa, including increased functional in vivohalf-life and/or increased plasma half-life, and/or increasedbioavailability and/or reduced sensitivity to proteolytic degradation.Consequently, medical treatment with a conjugate of the invention offersa number of advantages over the currently available rFVIIa compound,such as longer duration between injections.

Accordingly, in a first aspect, the invention relates to a conjugatecomprising at least one non-polypeptide moiety covalently attached to apolypeptide, wherein the amino acid sequence of the polypeptide differsfrom that of wild-type FVII or FVIIa shown in SEQ ID NO:1 in that atleast one amino acid residue comprising an attachment group for saidnon-polypeptide moiety has been introduced or removed.

In another aspect, the invention relates to a polypeptide, wherein theamino acid sequence of the polypeptide differs from that of wild-typeFVII or hFVIIa shown in SEQ ID NO:1 in that at least one amino acidresidue comprising an attachment group for a non-polypeptide moiety hasbeen introduced or removed. Such novel FVII polypeptides arecontemplated to be useful as such for therapeutic, diagnostic or otherpurposes, but find particular interest as intermediate products for thepreparation of a conjugate of the invention.

In further aspects, the invention relates to: a nucleotide sequenceencoding the polypeptide of the invention or the polypeptide part of theconjugate of the invention; an expression vector harbouring thenucleotide sequence of the invention; a host cell comprising thenucleotide sequence of the invention or the expression vector of theinvention.

In still further aspects, the invention relates to pharmaceuticalcompositions comprising the conjugate of the invention as well as tomethods for preparing and using such conjugates.

DETAILED DISCUSSION

Definitions

In the context of the present application and invention the followingdefinitions apply:

The term “conjugate” (or interchangeably “conjugated polypeptide”) isintended to indicate a heterogeneous (in the sense of composite orchimeric) molecule formed by the covalent attachment of one or morepolypeptide(s) to one or more non-polypeptide moieties such as polymermolecules, lipophilic compounds, sugar moieties or organic derivatizingagents. Preferably, the conjugate is soluble at relevant concentrationsand conditions, i.e., soluble in physiological fluids such as blood.Examples of conjugated polypeptides of the invention includeglycosylated and/or PEGylated polypeptides.

The term “covalent attachment” means that the polypeptide and thenon-polypeptide moiety are either directly covalently joined to oneanother, or else are indirectly covalently joined to one another throughan intervening moiety or moieties, such as a bridge, spacer, or linkagemoiety or moieties.

The term “non-conjugated polypeptide” can be used about the polypeptidepart of the conjugate.

When used herein, the term “non-polypeptide moiety” means a moleculethat is capable of conjugating to an attachment group of the polypeptideof the invention. Preferred examples of such molecules include polymermolecules, sugar moieties, lipophilic compounds, or organic derivatizingagents. When used in the context of a conjugate of the invention it willbe understood that the non-polypeptide moiety is linked to thepolypeptide part of the conjugate through an attachment group of thepolypeptide. As explained above, the non-polypeptide moiety can bedirectly covalently joined to the attachment group or it can beindirectly covalently joined to the attachment group through anintervening moiety or moieties, such as a bridge, spacer, or linkagemoiety or moieties.

The “polymer molecule” is a molecule formed by covalent linkage of twoor more monomers, wherein none of the monomers is an amino acid residue,except where the polymer is human albumin or another abundant plasmaprotein. The term “polymer” can be used interchangeably with the term“polymer molecule.” The term is intended to cover carbohydrate moleculesattached by in vitro glycosylation, i.e., a synthetic glycosylationperformed in vitro normally involving covalently linking a carbohydratemolecule to an attachment group of the polypeptide, optionally using across-linking agent.

Carbohydrate molecules attached by in vivo glycosylation, such as N- orO-glycosylation (as further described below) are referred to herein as a“sugar moiety.” Except where the number of non-polypeptide moieties,such as polymer molecule(s) or sugar moieties in the conjugate isexpressly indicated every reference to “a non-polypeptide moiety”contained in a conjugate or otherwise used in the present inventionshall be a reference to one or more non-polypeptide moieties, such aspolymer molecule(s) or sugar moieties.

The term “attachment group” is intended to indicate a functional groupof the polypeptide, in particular of an amino acid residue thereof or acarbohydrate moiety, capable of attaching a non-polypeptide moiety suchas a polymer molecule, a lipophilic molecule, a sugar moiety or anorganic derivatizing agent. Useful attachment groups and their matchingnon-polypeptide moieties are apparent from the table below.

Conjugation Attachment Examples of non- method/- group Amino acidpolypeptide moiety Activated PEG Reference —NH₂ N-terminal, Polymer,e.g., PEG, mPEG-SPA Shearwater Inc. Lys with amide or imine TresylatedmPEG Delgado et al, critical group reviews in Therapeutic Drug CarrierSystems 9(3, 4): 249-304 (1992) —COOH C-terminal, Polymer, e.g., PEG,mPEG-Hz Shearwater Inc. Asp, Glu with ester or amide group Carbohydratemoiety In vitro coupling —SH Cys Polymer, e.g., PEG, PEG-vinylsulphoneShearwater Inc. with disulfide, PEG-maleimide Delgado et al, criticalmaleimide or vinyl reviews in Therapeutic sulfone group Drug CarrierSystems Carbohydrate moiety In vitro coupling 9(3, 4): 249-304 (1992)—OH Ser, Thr, Sugar moiety In vivo O-linked Lys, OH— PEG with ester,glycosylation ether, carbamate, carbonate —CONH₂ Asn as part Sugarmoiety In vivo N- of an N- Polymer, e.g., PEG glycosylationglycosylation site Aromatic Phe, Tyr, Carbohydrate moiety In vitrocoupling residue Trp —CONH₂ Gln Carbohydrate moiety In vitro couplingYan and Wold, Biochemistry, 1984, Jul 31; 23(16): 3759-65 AldehydeOxidized Polymer, e.g., PEG, PEGylation Andresz et al., 1978, Ketoneoligo- PEG-hydrazide Makromol. Chem. 179:301, saccharide WO 92/16555, WO00/23114 Guanidino Arg Carbohydrate moiety In vitro coupling Lundbladand Noyes, Chimical Reagents for Protein Modification, CRC Press Inc.,Florida, USA Imidazole His Carbohydrate moiety In vitro coupling As forguanidine ring

For in vivo N-glycosylation, the term “attachment group” is used in anunconventional way to indicate the amino acid residues constituting aN-glycosylation site (with the sequence N-X-S/T/C, wherein X is anyamino acid residue except proline, N is asparagine and S/T/C is eitherserine, threonine or cysteine, preferably serine or threonine, and mostpreferably threonine). Although the asparagine residue of theN-glycosylation site is the one to which the sugar moiety is attachedduring glycosylation, such attachment cannot be achieved unless theother amino acid residues of the N-glycosylation site is present.

Accordingly, when the non-polypeptide moiety is a sugar moiety and theconjugation is to be achieved by N-glycosylation, the term “amino acidresidue comprising an attachment group for the non-polypeptide moiety”as used in connection with alterations of the amino acid sequence of thepolypeptide of interest is to be understood as meaning that one or moreamino acid residues constituting an N-glycosylation site are to bealtered in such a manner that either a functional N-glycosylation siteis introduced into the amino acid sequence or removed from saidsequence.

In the present application, amino acid names and atom names (e.g., CA,CB, CD, CG, SG, NZ, N, O, C, etc) are used as defined by the ProteinDataBank (PDB) (www.pdb.org) based on the IUPAC nomenclature (IUPACNomenclature and Symbolism for Amino Acids and Peptides (residue names,atom names, etc.), Eur. J. Biochem., 138, 9-37 (1984) together withtheir corrections in Eur. J. Biochem., 152, 1 (1985)).

The term “amino acid residue” is intended to indicate an amino acidresidue contained in the group consisting of alanine (Ala or A),cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E),phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H),isoleucine (Ile or I), lysine (Lys or K), leucine (Leu or L), methionine(Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gln orQ), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine(Val or V), tryptophan (Trp or W), and tyrosine (Tyr or Y) residues.

The terminology used for identifying amino acid positions is illustratedas follows: G124 indicates that position 124 is occupied by a glycineresidue in the amino acid sequence shown in SEQ ID NO:1. G124R indicatesthat the glycine residue of position 124 has been substituted with anarginine residue. Alternative substitutions are indicated with a “/”e.g., K32D/E means an amino acid sequence in which lysine in position 32is substituted with either aspartic acid or glutamic acid. Multiplesubstitutions are indicated with a “+,” e.g., K143N+N145S/T means anamino acid sequence which comprises a substitution of the lysine residuein position 143 with an asparagine residue and a substitution of theasparagine residue in position 145 with a serine or a threonine residue.The insertion of an additional amino acid residue, such as insertion ofan alanine residue after G124 is indicated by G124GA. A deletion of anamino acid residue is indicated by an asterix. For example, deletion ofa glycine in position 124 is indicated by G124*. Unless otherwiseindicated, the numbering of amino acid residues made herein is maderelative to the amino acid sequence of wild-type FVII/FVIIa shown in SEQID NO:1.

The term “differs from” as used in connection with specific mutations isintended to allow for additional differences being present apart fromthe specified amino acid difference. For instance, in addition to theremoval and/or introduction of amino acid residues comprising anattachment group for the non-polypeptide moiety the FVII or FVIIapolypeptide can comprise other substitutions that are not related tointroduction and/or removal of such amino acid residues. Thus, inaddition to the amino acid alterations disclosed herein aimed atremoving and/or introducing attachment sites for the non-polypeptidemoiety, it will be understood that the amino acid sequence of thepolypeptide of the invention can, if desired, contain other alterationsthat need not be related to introduction or removal of attachment sites,i.e., other substitutions, insertions or deletions. These can, forexample, include truncation of the N- and/or C-terminus by one or moreamino acid residues, or addition of one or more extra residues at the N-and/or C-terminus, e.g., addition of a methionine residue at theN-terminus as well as “conservative amino acid substitutions,” i.e.,substitutions performed within groups of amino acids with similarcharacteristics, e.g., small amino acids, acidic amino acids, polaramino acids, basic amino acids, hydrophobic amino acids and aromaticamino acids.

Preferred substitutions in the present invention can in particular beselected from the conservative substitution groups listed in the tablebelow.

1 Alanine (A) Glycine (G) Serine (S) Threonine (T) 2 Aspartic acid (D)Glutamic acid (E) 3 Asparagine (N) Glutamine (Q) 4 Arginine (R)Histidine (H) Lysine (K) 5 Isoleucine (I) Leucine (L) Methionine (M)Valine (V) 6 Phenylalanine (F) Tyrosine (Y) Tryptophan (W)

The terms “mutation” and “substitution” are used interchangeably herein.

The term “nucleotide sequence” is intended to indicate a consecutivestretch of two or more nucleotide molecules. The nucleotide sequence canbe of genomic, cDNA, RNA, semisynthetic, synthetic origin, or anycombinations thereof.

The term “polymerase chain reaction” or “PCR” generally refers to amethod for amplification of a desired nucleotide sequence in vitro, asdescribed, for example, in U.S. Pat. No. 4,683,195. In general, the PCRmethod involves repeated cycles of primer extension synthesis, usingoligonucleotide primers capable of hybridising preferentially to atemplate nucleic acid.

“Cell,” “host cell,” “cell line” and “cell culture” are usedinterchangeably herein and all such terms should be understood toinclude progeny resulting from growth or culturing of a cell.“Transformation” and “transfection” are used interchangeably to refer tothe process of introducing DNA into a cell.

“Operably linked” refers to the covalent joining of two or morenucleotide sequences, by means of enzymatic ligation or otherwise, in aconfiguration relative to one another such that the normal function ofthe sequences can be performed. For example, the nucleotide sequenceencoding a presequence or secretory leader is operably linked to anucleotide sequence coding for a polypeptide if it is expressed as apreprotein that participates in the secretion of the polypeptide: apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the sequence; a ribosome binding site isoperably linked to a coding sequence if it is positioned so as tofacilitate translation. Generally, “operably linked” means that thenucleotide sequences being linked are contiguous and, in the case of asecretory leader, contiguous and in reading phase. Linking isaccomplished by ligation at convenient restriction sites. If such sitesdo not exist, then synthetic oligonucleotide adaptors or linkers areused, in conjunction with standard recombinant DNA methods.

The term “introduce” refers to introduction of an amino acid residuecomprising an attachment group for a non-polypeptide moiety, inparticular by substitution of an existing amino acid residue, oralternatively by insertion of an additional amino acid residue.

The term “remove” refers to removal of an amino acid residue comprisingan attachment group for a non-polypeptide moiety, in particular bysubstitution of the amino acid residue to be removed by another aminoacid residue, or alternatively by deletion (without substitution) of theamino acid residue to be removed

The term “FVII” or “FVII polypeptide” refers to a FVII molecule providedin single chain form.

The term “FVIIa” or “FVIIa polypeptide” refers to a FVIIa moleculeprovided in its activated two-chain form, wherein the peptide bondbetween R152 and I153 of the single-chain form has been cleaved. Whenthe amino acid sequence of SEQ ID NO; 1 is used herein to describe theamino acid sequence of FVIIa it will be understood to that one of thechains comprises amino acid residues 1-152, the other chain amino acidresidues 153-406.

The terms “rFVII” and “rFVIIa” refer to FVII and FVIIa moleculesproduced by recombinant techniques, respectively.

The terms “hFVII” and “hFVIIa” refer to wild-type human FVII and FVIIa,respectively.

The term “catalytic site” is used to mean the catalytic triad consistingof S344, D242 and H193 of the FVII polypeptide.

The term “active FVIIa,” “active FVIIa polypeptide,” “active FVIIaconjugate” or “active conjugate” is used to mean a FVIIa polypeptide orconjugate that possesses at least 10% of the catalytic activity ofwild-type hFVIIa. Catalytic acitivity, as used herein, can suitably bedetermined in the assay described in the section entitled Method ofmeasuring the catalytic activity or in the assay entitled Method ofmeasuring low levels of catalytic activity (see the Materials andMethods section herein). Preferably, an active conjugate has at least15%, such as at least 20%, e.g., at least 25%, more preferably at least30%, such as at least 40%, most preferably at least 50%, e.g., at least60% of the catalytic acitivity of wild-type hFVIIa, when tested in theassays described above.

Preferably, an active conjugate is able to bind to tissue factor andfurther activate plasma factor X and/or IX. Thus, in a preferredembodiment, the active FVIIa polypeptide or a conjugate thereof has aclotting activity of at least 25% as compared to wild type FVIIa, suchas a clotting activity of at least 50% as compared to wild type FVIIa,e.g., a clotting activity of at least 75% as compared to wild type FVII.Thus, the clotting activity of the active FVIIa polypeptide or conjugatethereof is preferably in the range of 25-200% as compared to the wildtype FVIIa. In particular, it is preferred that the FVIIa polypeptide orconjugate has a clotting activity in the range of 30-150% as compared towild type FVIIa, such as a clotting activity in the range of 30-100% ascompared to wild type FVIIa. The clotting activity can be determined byany method known in the art as further discussed in the Materials andMethods section hereinafter. It is particularly preferred, however, thatthe clotting activity is determined in accordance with the methoddescribed in the section entitled “Method of measuring the clottingactivity” (see the Materials and Method section).

The term “immunogenicity” as used in connection with a given substanceis intended to indicate the ability of the substance to induce aresponse from the immune system. The immune response can be a cell orantibody mediated response (see, e.g., Roitt: Essential Immunology(8^(th) Edition, Blackwell) for further definition of immunogenicity).Normally, reduced antibody reactivity will be an indication of reducedimmunogenicity. The reduced immunogenicity can be determined by use ofany suitable method known in the art, e.g., in vivo or in vitro.

The term “inactive FVIIa,” “inactive FVIIa polypeptide,” “inactive FVIIaconjugate” or “inactive conjugate” is used to mean a FVIIa polypeptideor conjugate that possesses less than 10% of the catalytic activity ofwild-type hFVIIa. Catalytic acitivity, as used herein, can suitably bedetermined in the assay described in the section entitled Method ofmeasuring the catalytic activity or in the assay entitled Method ofmeasuring low levels of catalytic activity (see the Materials andMethods section herein). Preferably, an inactive conjugate has less than8%, such as less than 6%, e.g., less than 5%, more preferably less than4%, such as less than 3%, most preferably less than 2%, e.g., less than1% of the catalytic acitivity of wild-type hFVIIa, when tested in theassays described above.

Typically, an inactive conjugate has significantly reduced in vitro orin vivo clotting activity as compared to wild-type hFVIIa. The inactiveFVII or FVIIa polypeptide or conjugate can be capable of competing withwild-type FVII or FVIIa for binding tissue factor, thereby inhibitingclotting activity. Preferably, the inactive FVII or FVIIa polypeptide orconjugate has less than 1% clotting activity compared to wild-type hFVIIor hFVIIa. More preferably the inactive FVII or FVIIa polypeptide orconjugate has less than 0.05% clotting activity compared to wild typehFVII or hFVIIa. Most preferably the inactive FVII or FVIIa polypeptideor conjugate has less than 0.01% clotting activity as compared to wildtype hFVII or hFVIIa. In a similar way as described above, the clottingactivity can be determined by any method known in the art as furtherdiscussed in the Materials and Method section hereinafter, but ispreferably determined in accordance with the method described in thesection entitled “Method of measuring the clotting activity.”

The term “functional in vivo half-life” is used in its normal meaning,i.e., the time at which 50% of the biological activity of thepolypeptide or conjugate is still present in the body/target organ, orthe time at which the activity of the polypeptide or conjugate is 50% ofthe initial value. As an alternative to determining functional in vivohalf-life, “serum half-life” can be determined, i.e., the time at which50% of the polypeptide or conjugate molecules circulate in the plasma orbloodstream prior to being cleared. Determination of serum half-life isoften more simple than determining the functional in vivo half-life andthe magnitude of serum half-life is usually a good indication of themagnitude of functional in vivo half-life. Alternatively terms to serumhalf-life include “plasma half-life,” “circulating half-life,” “serumclearance,” “plasma clearance” and “clearance half-life.” Thepolypeptide or conjugate is cleared by the action of one or more of thereticuloendothelial systems (RES), kidney, spleen or liver, by tissuefactor, SEC receptor or other receptor mediated elimination, or byspecific or unspecific proteolysis. Normally, clearance depends on size(relative to the cutoff for glomerular filtration), charge, attachedcarbohydrate chains, and the presence of cellular receptors for theprotein. The functionality to be retained is normally selected fromprocoagulant, proteolytic or receptor binding activity. The functionalin vivo half-life and the serum half-life can be determined by anysuitable method known in the art as further discussed in the MaterialsMethods section below.

The term “increased” as used about the functional in vivo half-life orplasma half-life is used to indicate that the relevant half-life of theconjugate or polypeptide is statistically significantly increasedrelative to that of a reference molecule, such as a non-conjugatedrFVIIa (e.g., NovoSeven®) as determined under comparable conditions. Forinstance, the relevant half-life can increased by at least about 25%,such as by at least about 50%, e.g., by at least about 100%, 150%, 200%,250%, 300%, 500% or 1000%.

The term “renal clearance” is used in its normal meaning to indicate anyclearance taking place by the kidneys, e.g., by glomerular filtration,tubular excretion or degradation in the tubular cells. Renal clearancedepends on physical characteristics of the conjugate, including size(diameter), hydrodynamic volume, symmetry, shape/rigidity, and charge.Normally, a molecular weight of about 67 kDa is considered to be acut-off-value for renal clearance. Renal clearance can be established byany suitable assay, e.g., an established in vivo assay. Typically, renalclearance is determined by administering a labelled (e.g., radiolabelledor fluorescence labelled) polypeptide conjugate to a patient andmeasuring the label activity in urine collected from the patient.Reduced renal clearance is determined relative to a correspondingreference polypeptide, e.g., the corresponding non-conjugatedpolypeptide, a non-conjugated corresponding wild-type polypeptide oranother conjugated polypeptide (such as a conjugated polypeptide notaccording to the invention), under comparable conditions. Preferably,the renal clearance rate of the conjugate is reduced by at least 50%,preferably by at least 75%, and most preferably by at least 90% comparedto a relevant reference polypeptide.

The ability of the conjugates of the invention to exhibit a reducedsensitivity to proteolytic degradation is of utmost importance;Compositions comprising degradation products will typically have lessspecific activity as compared to compositions in which none or only aminor part of the conjugate has been degraded. Furthermore, a content ofnon-physiological degradation products in the composition to beadministered can trigger the immune system of the patient.

The term “reduced sensitivity to proteolytic degradation” is primarilyintended to mean that the conjugate has reduced sensitivity toproteolytic degradation in comparison to non-conjugated wild type FVIIaas determined under comparable conditions. Preferably, the proteolyticdegradation is reduced by at least 10%, such as at least 25% (e.g., by10-25%), more preferably by at least 35%, such as at least 50%, (e.g.,by 10-50%, such as 25-50%) even more preferably by at least 60%, such asby at least 75% or even at least 90%. Most preferably, the proteolyticdegradation is reduced by 100%. Thus, preferably the conjugate of theinvention is subjected to proteolytic degradation to a lesser extentthan wild-type FVIIa, i.e., compared to non-conjugated wild type FVIIathe proteolytic degradation of the conjugate of the invention ispreferably reduced by 10-100%, such as by 25-100%, more preferably by50-100%, and most preferably by 75-100%.

The present inventors have developed a suitable preliminary in vitrotest, which can be employed in the assessment of whether such conjugatespossess reduced sensitivity to proteolytic cleavage (reducedautoproteolysis). Thus, in a preferred embodiment, of the invention, theconjugate of the invention has a reduced sensitivity to proteolyticdegradation (as defined above) as compared to wild type FVIIa whendetermined by the method described in the section entitled “Measurementof reduced sensitivity to proteolytic degradation,” when determined bythe method described in the section entitled Method of measuring thecatalytic activity or when determined by the method described in thesection entitled Method of measuring low levels of catalytic activity(see the Materials and Methods section herein).

The term “parent FVII” or “parent polypeptide” is intended to indicatethe molecule to be modified in accordance with the present invention. Atypical parent FVII is the hFVII or hFVIIa (including rFVIIa(NovoSeven®)) with the amino acid sequence shown in SEQ ID NO:1.

A “variant” is a polypeptide, which differs in one or more amino acidresidues from a parent polypeptide, normally in 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14 or 15 amino acid residues.

Conjugate of the Invention

The conjugates of the invention are the result of a generally newstrategy for developing improved FVII or FVIIa molecules. Morespecifically, by removing and/or introducing an amino acid residuecomprising an attachment group for the non-polypeptide moiety it ispossible to specifically adapt the polypeptide so as to make themolecule more susceptible to conjugation to the non-polypeptide moietyof choice, to optimize the conjugation pattern (e.g., to ensure anoptimal distribution and number of non-polypeptide moieties on thesurface of the FVII or FVIIa molecule and to ensure that only theattachment groups intended to be conjugated is present in the molecule)and thereby obtain a new conjugate molecule, which has or has not FVIIactivity and in addition one or more improved properties as compared toFVII and FVIIa molecules available today. For instance, when the totalnumber of amino acid residues comprising an attachment group for thenon-polypeptide of choice is increased or decreased to an optimizedlevel, the renal clearance of the conjugate is typically significantlyreduced due to the altered shape, size and/or charge of the moleculeachieved by the conjugation.

In preferred embodiments of the present invention more than one aminoacid residue of the FVII or FVIIa polypeptide is altered, e.g., thealteration embraces removal as well as introduction of amino acidresidues comprising an attachment group for the non-polypeptide moietyof choice. In addition to the removal and/or introduction of amino acidresidues the polypeptide can comprise other substitutions orglycosylations that are not related to introduction and/or removal ofamino acid residues comprising an attachment group for thenon-polypeptide moiety. Also, the polypeptide can be attached, e.g., toa serine proteinase inhibitor to inhibit the catalytic site of thepolypeptide.

The amino acid residue comprising an attachment group for anon-polypeptide moiety, either it be removed or introduced, is selectedon the basis of the nature of the non-polypeptide moiety of choice and,in most instances, on the basis of the method in which conjugationbetween the polypeptide and the non-polypeptide moiety is to beachieved. For instance, when the non-polypeptide moiety is a polymermolecule such as a polyethylene glycol or polyalkylene oxide derivedmolecule amino acid residues comprising an attachment group can beselected from the group consisting of lysine, cysteine, aspartic acid,glutamic acid, histidine, and tyrosine, preferably cysteine and lysine,in particular lysine.

Whenever an attachment group for a non-polypeptide moiety is to beintroduced into or removed from the FVII or FVIIa polypeptide inaccordance with the present invention, the position of the polypeptideto be modified is preferably located at the surface of the poly-peptide,and more preferably occupied by an amino acid residue which has morethan 25% of its side chain exposed to the solvent, preferably more than50% of its side chain exposed to the solvent. Such positions have beenidentified on the basis of an analysis of a 3D structure of the humanFVII or FVIIa molecule as described in the Materials and Methods sectionherein. Furthermore, the position is preferably selected from a part ofthe FVII molecule that is located outside a tissue factor binding siteregion and/or an active site region. These regions are identified in theMaterials and Methods section hereinafter. It should be emphasized,however, that in certain situations, e.g., in case an inactivatedconjugate is desired, it can be advantageous to perform modifications inor close to such regions. For example, it is contemplated that one ormore attachment groups for the non-polypeptide moieties, such asattachment groups for in vivo N-glycosylation sites, can advantageouslybe inserted in the active site region or at the ridge of the active sitebinding cleft of the FVII molecule. The active site region and the ridgeof the active site binding cleft are defined in the Materials andMethods section herein and is constituted by the following residues:

I153, Q167, V168, L169, L170, L171, Q176, L177, C178, G179, G180, T181,V188, V189, S190, A191, A192, H193, C194, F195, D196, K197, I198, W201,V228, I229 , I230, P231, S232, T233, Y234, V235, P236, G237, T238, T239,N240, I241, D242, I243, A244, L245, L246, V281, S282, G283, W284, G285,Q286, T293, T324, E325, Y326, M327, F328, D338, S339, C340, K341, G342,D343, S344, G345, G346, P347, H348, L358, T359, G360, I361, V362, S363,W364, G365, C368, V376, Y377, T378, R379, V380, Q382, Y383, W386, L387,L400 and F405 (active site region); and N173, A175, K199, N200, N203,D289, R290, G291, A292, P321 and T370 (the ridge of the active sitebinding cleft).

In order to determine an optimal distribution of attachment groups, thedistance between amino acid residues located at the surface of the FVIIor FVIIa molecule is calculated on the basis of a 3D structure of thepolypeptide. More specifically, the distance between the CB's of theamino acid residues comprising such attachment groups, or the distancebetween the functional group (NZ for lysine, CG for aspartic acid, CDfor glutamic acid, SG for cysteine) of one and the CB of another aminoacid residue comprising an attachment group are determined. In case ofglycine, CA is used instead of CB. In the FVII or FVIIa polypeptide partof a conjugate of the invention, any of said distances is preferablymore than 8 Å, in particular more than 10 Å in order to avoid or reduceheterogeneous conjugation.

In case of removal of an attachment group, the relevant amino acidresidue comprising such group and occupying a position as defined aboveis preferably substituted with a different amino acid residue that doesnot comprise an attachment group for the non-polypeptide moiety inquestion. Normally, the amino acid residue to be removed is one to whichconjugation is disadvantageous, e.g., an amino acid residue located ator near a functional site of the polypeptide (since conjugation at sucha site can result in inactivation or reduced FVII or FVIIa activity ofthe resulting conjugate due to impaired receptor recognition). In thepresent context the term “functional site” is intended to indicate oneor more amino acid residues which is/are essential for or otherwiseinvolved in the function or performance of FVII or FVIIa. Such aminoacid residues are a part of the functional site. The functional site canbe determined by methods known in the art and is preferably identifiedby analysis of a structure of the FVIIa-tissue factor complex (SeeBanner et al., Nature 1996; 380:41-46).

In case of introduction of an attachment group, an amino acid residuecomprising such group is introduced into the position, preferably bysubstitution of the amino acid residue occupying such position.

The exact number of attachment groups present and available forconjugation in the FVII or FVIIa polypeptide is dependent on the effectdesired to be achieved by the conjugation. The effect to be obtained is,e.g., dependent on the nature and degree of conjugation (e.g., theidentity of the non-polypeptide moiety, the number of non-polypeptidemoieties desirable or possible to conjugate to the polypeptide, wherethey should be conjugated or where conjugation should be avoided, etc.).

Functional in vivo half-life is i.a. dependent on the molecular weightof the conjugate, and the number of attachment groups needed forproviding increased half-life thus depends on the molecular weight ofthe non-polypeptide moiety in question. In one embodiment, the conjugateof the invention has a molecular weight of at least 67 kDa, inparticular at least 70 kDa, e.g., as measured by SDS-PAGE according toLaemmli, U.K., Nature Vol 227 (1970), p680-85. FVII has a molecularweight of about 53 kDa, and therefore additional 10-20 kDa is requiredto obtain the desired effect. This can, e.g., be provided by conjugating2-4 10 kDa PEG molecules or as otherwise described herein.

In order to avoid too much disruption of the structure and function ofthe parent molecule the polypeptide part of the conjugate will typicallyhave an amino acid sequence having more than 90% identity with SEQ IDNO:1, preferably more than 95%, such as more than 96%. In particular,the polypeptide part of the conjugate will typically have an amino acidsequence having more than 97% identity with SEQ ID NO:1, such as morethan 98%, more than 99%, more than 99.25%, more than 99.25% or more than99.5%.

Amino acid sequence homology/identity is conveniently determined fromaligned sequences, using e.g., the ClustalW program, version 1.8, June1999, using default parameters (Thompson et al., 1994, ClustalW:Improving the sensitivity of progressive multiple sequence alignmentthrough sequence weighting, position-specific gap penalties and weightmatrix choice, Nucleic Acids Research, 22: 4673-4680) or from the PFAMfamilies database version 4.0 (http://pfam.wustl.edu/) (Nucleic AcidsRes. 1999 Jan. 1; 27(1):260-2) by use of GENEDOC version 2.5 (Nicholas,K. B., Nicholas H. B. Jr., and Deerfield, D. W. II. 1997 GeneDoc:Analysis and Visualization of Genetic Variation, EMBNEW.NEWS 4:14;Nicholas, K. B. and Nicholas H. B. Jr. 1997 GeneDoc: Analysis andVisualization of Genetic Variation).

Stated differently, the total number of amino acid residues to bealtered in accordance with the present invention (as compared to theamino acid sequence shown in SEQ ID NO:1) will typically not exceed 15.Preferably, the FVII or FVIIa polypeptide part of the conjugate of theinvention or the polypeptide of the invention comprises an amino acidsequence differing in 1-15 amino acid residues from the amino acidsequence shown in SEQ ID NO:1, typically in 1-10 or in 2-10 amino acidresidues, e.g., in 1-8 or in 2-8 amino acid residues, such as in 3-7 orin 4-6 amino acid residues from the amino acid sequence shown in SEQ IDNO:1. Thus, normally the polypeptide part of the conjugate or thepolypeptide of the invention comprises an amino acid sequence whichdiffers from the amino acid sequence shown in SEQ ID NO:1 in at the most15 amino acid residues (such as 15 amino acid residues), in at the most14 amino acid residues (such as 14 amino acid residues), in at the most13 amino acid residues (e.g., 13 amino acid residues), in at the most 12amino acid residues (such as 12 amino acid residues), in at the most 11amino acid residues (such as 11 amino acid residues), in at the most 10amino acid residues (e.g., 10 amino acid residues), in at the most 9amino acid residues (such as 9 amino acid residues), in at the most 8amino acid residues (such as 8 amino acid residues), in at the most 7amino acid residues (such as 7 amino acid residues), in at the most 6amino acid residues (such as 6 amino acid residues), in at the most 5amino acid residues (such as 5 amino acid residues), in at the most 4amino acid residues (such as 4 amino acid residues), in at the most 3amino acid residues (such as 3 amino acid residues) or in at the most 2amino acid residues (such as 2 amino acid residues).

Analogously, the conjugate of the invention typically contains, e.g.,1-15 non-polypeptide moieties, typically 1-10 non-polypeptide moietiesor 2-10 non-polypeptide moieties, such as 1-8 or 2-8 non-polypeptidemoieties, e.g., 1-6, 1-4, 3-7 or 4-6 non-polypeptide moieties.

Preferably, the conjugate of the invention has one or more of thefollowing improved properties: Increased functional in vivo half-life,increased plasma half-life, reduced renal clearance and reducedsensitivity to proteolytic degradation as compared to rFVIIa (e.g.NovoSeven®).

It is known from, inter alia, WO 88/10295 that proteolytic degradationof FVII/FVIIa primarily takes place at various proteolytic sites in themolecule, namely at the positions K32, K38, I42, Y44, K143, R290, R315,K341, R392, R396 and R402 (or rather between the positions K32-D33,K38-L39, I42-S43, Y44-S45, K143-R144, R290-G291, R315-K316, K341-G342,R392-S393, R396-P397 and R402-A403). Thus, one preferred strategy forincreasing e.g., the functional in vivo half-life, the increased plasmahalf-life or for reducing the sensitivity to proteolytic degradation isto modify the parent polypeptide at and/or around one or more of theseproteolytic degradation sites, e.g., by introducing non-polypeptidemoieties at and/or around these sites. Thus, in a preferred embodiment,of the invention, an attachment group has been introduced in one or moreof the above-mentioned identified positions or at position −4, −3, −2,−1, 1, 2, 3, 4, preferably position −2, −1, 1, 2, such as position −1,1, relative to the position directly involved in proteolyticdegradation.

More specifically, it is preferred that a non-naturally occurring invivo glycosylation site, in particular a non-naturally occurring in vivoN-glycosylation site (see further below) has been introduced inpositions selected from the group consisitng of 28-48, 139-147, 286-294,311-319, 338-345, and 388-406. In particular, it is preferred that thein vivo glycosylation site, such as an in vivo N-glycosylation site (seefurther below) has been introduced in positions selected from the groupconsisiting of 30-34, 36-46, 141-144, 288-292, 313-317, 341-343, 390-398and 400-404. More preferably, the in vivo glycosylation site, such as anin vivo N-glycosylation site (see further below) has been introduced inpositions selected from the group consisiting of 31-33 (e.g., positions31, 32 or 33), 37-39 (e.g., positions 37, 38 or 39), 41-46 (e.g.,positions 41, 42, 43, 44, 45 or 46), 142-144 (e.g., positions 142, 143or 144), 289-291 (e.g., positions 289, 290 or 291), 314-316, (e.g.,positions 314, 315 or 316), 341-342 (e.g., positions 341, 342 or 343),391-393 (e.g., positions 391, 392 or 393), 395-397 (e.g., positions 395,396 or 397) and 401-403 (e.g., positions 401, 402 or 403). Theintroduction is preferably performed by susbstitution.

Conjugate of the Invention, wherein the Non-Polypeptide Moiety is aMolecule that Has Lysine as an Attachment Group

In another embodiment of the invention, the non-polypeptide moiety haslysine as an attachment group, i.e., the conjugate of the invention isone which comprises at least one non-polypeptide moiety covalentlyattached to a polypeptide, wherein the amino acid sequence of thepolypeptide differs from that of wild-type FVII or FVIIa shown in SEQ IDNO: 1 in that at least one lysine residue has been introduced orremoved.

FVII/FVIIa contains 17 lysine residues. Three lysine residues (K18, K62and K85) are located in the tissue factor binding domain and two lysineresidues (K197 and K341) are located in the active site region.

Due to the relative high amount of lysine residues in the parentpolypeptide it is envisaged that at least one lysine residue shouldpreferably be removed, in particular by substitution of a lysine residuewith a non-lysine residue, in order to avoid excessive conjugation tonon-polypeptide moieties.

Thus, in one embodiment, the amino acid sequence of the FVII or FVIIapolypeptide part of the conjugate differs from that shown in SEQ ID NO:1in that at least one lysine residue, such as 1-15 lysine residues, inparticular 1-10, 1-6 or 2-4 lysine residues, has been removed,preferably by substitution. For example, the lysine residue(s) to beremoved, preferably by substitution, is selected from the groupconsisting of K18, K32, K38, K62, K85, K109, K137, K143, K148, K157,K161, K197, K199, K316, K337, K341, K389 and combinations thereof. Inparticular, it is preferred to remove one or more lysine residues, whichconstitute part of tissue factor binding site and/or the active siteregion, i.e., the residues K18, K62, K85, K197, K341 or combinationsthereof. The lysine residue can be substituted with any other amino acidresidue, but is preferably substituted with R, Q, N or H, morepreferably R.

In another embodiment, the amino acid sequence of the FVII or FVIIapolypeptide part of the conjugate differs from that shown in SEQ ID NO:1in that at least one lysine residue, such as 1-15 lysine residues, inparticular 1-10, 1-6 or 24 lysine residues, has been introduced,preferably by substitution. It will be understood that it isparticularly preferred that at least one of the lysines residues, whichare introduced at a predetermined site in the parent molecule, iscombined with at least one of the above-mentioned removals of lysineresidues. Thus, in a preferred embodiment of the invention, the aminoacid sequence of the FVII or FVIIa polypeptide part of the conjugatediffers from that shown in SEQ ID NO:1 in that at least one lysineresidue has been removed and at least one lysine residue has beenintroduced.

Examples of positions, wherein lysine residues can be introducedinclude, but is not limited to, positions at or in the vicinity of theproteolytic degradation sites described above. Thus, in a preferredembodiment, the substitution of a non-lysine residue with a lysineresidue is selected from the group consisting of I42K, Y44K, L288K,D289K, R290K, G291K, A292K, T293K, Q313K, S314K, R315K, V317K, L390K,M391K, R392K, S393K, E394K, P395K, R396K, P397K, G398K, V399K, L400K,L401K, R402K, A403K, P404K, F405K and combinations thereof, inparticular selected from the group consisting of R290K, R315K, R392K,R396K, R402K and combinations thereof.

While the non-polypeptide moiety of the conjugate according to thisaspect of the invention can be any molecule which, when using the givenconjugation method has lysine as an attachment group it is preferredthat the non-polypeptide moiety is a polymer molecule. The polymermolecule can be any of the molecules mentioned in the section entitled“Conjugation to a polymer molecule,” but is preferably selected from thegroup consisting of linear or branched polyethylene glycol or anotherpolyalkylene oxide. Examples of preferred polymer molecules are, e.g.,SS-PEG, NPC-PEG, aldehyde-PEG, mPEG-SPA, mPEG-SCM, mPEG-BTC fromShearwater Polymers, Inc, SC-PEG from Enzon, Inc., tresylated mPEG asdescribed in U.S. Pat. No. 5,880,255, oroxycarbonyl-oxy-N-dicarboxyimide-PEG (U.S. Pat. No. 5,122,614).

Normally, for conjugation to a lysine residue the non-polypeptide moietyhas a molecular weight of from about 5 to about 20 kDa, such as fromabout 5 to about 10 kDa, e.g., about 5 kDa or about 10 kDa

It will be understood that any of the amino acid changes, in particularsubstitutions, specified in this section can be combined with any of theamino acid changes, preferably substitutions specified in the othersections herein disclosing specific amino acid modifications, includingintroduction and/or removal of glycosylation sites.

Conjugate of the Invention wherein the Non-Polypeptide Moiety is aMolecule that Has Cysteine as an Attachment Group

In a further embodiment of the invention, the non-polypeptide moiety hascysteine as an attachment group, i.e., the conjugate of the invention isone which comprises at least one non-polypeptide moiety covalentlyattached to a polypeptide, wherein the amino acid sequence of thepolypeptide differs from that of wild-type FVII or FVIIa shown in SEQ IDNO:1 in that at least one cysteine residue has been introduced orremoved.

FVII/FVIIa contains 24 cysteine residues and disulfide bridges areestablished between the following cysteine residues: C17 and C22, C50and C61, C55 and C70, C72 and C81, C91 and C102, C98 and C112, C114 andC127, C135 and C262, C159 and C164, C178 and C194, C310 and C329, andbetween C340 and C368.

In a further interesting embodiment, the amino acid sequence of the FVIIor FVIIa polypeptide differs from that shown in SEQ ID NO:1 in that atleast one cysteine residue, such as 1-15 cysteine residues, inparticular 1-10, 1-6 or 2-4 cysteine residues, has been introduced,preferably by substitution.

Examples of positions, wherein cysteine residues can be introducedinclude, but is not limited to, positions at or in the vicinity of theproteolytic degradation sites described above.

Thus, in an interesting embodiment of the invention, the lysineresidue(s) to be introduced, preferably by substitution, is selectedfrom the group consisting of I30C, K32C, D33C, A34C, T37C, K38C, W41C,Y44C, S45C, D46C, L141C, E142C, K143C, R144C, L288C, D289C, R290C,G291C, A292C, S314C, R315C, K316C, V317C, L390C, M391C, R392C, S393C,E394C, P395C, R396C, P397C, G398C, V399C, L401C, R402C, A403C, P404C andcombinations thereof, in particular selected from the group consistingof K32C, Y44C, K143C, R290C, R315C, K341C, R392C, R396C, R402C andcombinations thereof.

In a further embodiment of the invention, the cysteine residue(s) is/areintroduced into a position that in wildtype hFVII is occupied by athreonine or serine residue having at least 25% of its side chainexposed to the surface. For instance, in the FVII or FVIIa polypeptide acysteine residue is introduced, preferably by substitution, into atleast one position selected from the group consisting of S12, S23, S43,S45, S52, S53, S60, S67, T83, S103, T106, T108, S111, S119, S126, T128,T130, S147, T185, S214, S222, S232, T233, T238, T239 , T255, T267, T293,T307, S320, T324, S333, S336, T370 and S393. Even more preferably thecysteine residue is introduced into at least one position of hFVIIcontaining an S residue, the position being selected from the groupconsisting of S12, S23, S43, S45, S52, S53, S60, S67, S103, S111, S119,S126, S147, S214, S222, S232, S320, S333, S336 and S393.

In a further embodiment, the cysteine residue(s) is/are introduced intoa position that in wildtype hFVII is occupied by a threonine or serineresidue having at least 50% of its side chain exposed to the surface.For instance, in the FVII or FVIIa polypeptide a cysteine residue isintroduced, preferably by substitution, into at least one positionselected from the group consisting of S23, S43, S52, S53, S60, S67,T106, T108, S111, S119, S147, S214, T238, T267, and T293, even morepreferably a position selected from the group consisting of S23, S43,S52, S53, S60, S67, S111, S119, S147 and S214.

In a still further embodiment, a cysteine residue is introduced into atleast one position selected from any of the above-mentioned positions,which is not located in an active site region. Preferably, the positionis one occupied by a T or an S residue. As an example, the FVIIpolypeptide comprises a cysteine residue introduced into at least oneposition selected from the group consisting of S12, S23, S43, S45, S52,S53, S60, S67, T83, S103, T106, T108, S111, S119, S126, T128, T130,S147, T185, S214, S222, T255, T267, T307, S320, S333, S336, T370 andS393 (having more than 25% of its side chain exposed to the surface), inparticular selected from the group consisting of S12, S23, S43, S45,S52, S53, S60, S67, S103, S111, S119, S126, S147, S214, S222, S320,S333, S336 and S393 (occupied by S residue), and more preferably fromthe group consisting of S23, S43, S52, S53, S60, S67, T106, T108, S111,S119, S147, S214 and T267 (having more than 50% of its side chainexposed to the surface), in particular from the group consisting of S23,S43, S52, S53, S60, S67, S111, S119, S147 and S214 (occupied by an Sresidue).

In an even further embodiment, a cysteine residue is introduced into atleast one position selected from any of the above lists, which is notlocated in a tissue factor binding site region. Preferably, the positionis one occupied by a T or an S residue. As an example, the FVIIpolypeptide comprises a cysteine residue introduced into at least oneposition selected from the group consisting of S12, S23, S45, S52, S53,S67, T83, S103, T106, T108, S111, S119, S126, T128, T130, S147, T185,S214, S222, S232, T233, T238, T239, T255, T267, T293, S320, T324, S333,S336, T370 and S393 (having more than 25% of its side chain exposed tothe surface), in particular selected from the group consisting of S12,S23, S45, S52, S53, S67, S103, S111, S119, S126, S147, S214, S222, S232,S320, S333, S336 and S393 (occupied by S residue), and more preferablyfrom the group consisting of S23, S52, S53, S67, T106, T108, S111, S119,S147, S214, T238, T267 and T293 (having more than 50% of its side chainexposed to the surface), in particular from the group consisting of S23,S52, S53, S67, S111, S119, S147 and S214 (occupied by an S residue).

In a still further embodiment, a cysteine residue is introduced into atleast one position selected from any of the above lists, which isneither located in a tissue factor binding site region nor in an activesite region. Preferably, the position is one occupied by a T or an Sresidue. As an example, the FVII polypeptide comprises a cysteineresidue introduced into at least one position selected from the groupconsisting of S12, S23, S45, S52, S53, S67, T83, S103, T106, T108, S111,S119, S126, T128, T130, S147, T185, S214, S222, T255, T267, S320, S333,S336, T370 and S393 (having more than 25% of its side chain exposed tothe surface), in particular selected from the group consisting of S12,S23, S45, S52, S53, S67, S103, S111, S119, S126, S147, S214, S222, S320,S333, S336 and S393 (occupied by S residue), and more preferably fromthe group consisting of S23, S52, S53, S67, T106, T108, S111, S119,S147, S214 and T267 (having more than 50% of its side chain exposed tothe surface), in particular from the group consisting of S23, S52, S53,S67, S111, S119, S147 and S214 (occupied by an S residue).

While the non-polypeptide moiety of the conjugate according to thisaspect of the invention can be any molecule which, when using the givenconjugation method has cysteine as an attachment group it is preferredthat the non-polypeptide moiety is a polymer molecule. The polymermolecule can be any of the molecules mentioned in the section entitled“Conjugation to a polymer molecule,” but is preferably selected from thegroup consisting of linear or branched polyethylene glycol or anotherpolyalkylene oxide. In another embodiment, the polymer molecule is PEG,such as VS-PEG. The conjugation between the polypeptide and the polymercan be achieved in any suitable manner, e.g., as described in thesection entitled “Conjugation to a polymer molecule,” e.g., in using aone step method or in the stepwise manner referred to in said section.When the FVII or FVIIa polypeptide comprises only one conjugatablecysteine residue, this is preferably conjugated to a non-polypeptidemoiety with a molecular weight of from about 5 kDa to about 20 kDa,e.g., from about 10 kDa to about 20 kDa, such as a molecular weight ofabout 5 kDa, about 10 kDa, about 12 kDa, about 15 kDa or about 20 kDa,either directly conjugated or indirectly through a low molecular weightpolymer (as disclosed in WO 99/55377). When the conjugate comprises twoor more conjugatable cysteine residue, normally each of thenon-polypeptide moieties has a molecular weight of from about 5 to about10 kDa, such as about 5 kDa or about 10 kDa.

It will be understood that any of the amino acid changes, in particularsubstitutions, specified in this section can be combined with any of theamino acid changes, preferably substitutions specified in the othersections herein disclosing specific amino acid modifications, includingintroduction and/or removal of glycosylation sites.

Conjugate of the Invention wherein the Non-Polypeptide Moiety is aMolecule that Has Aspartic Acid or Glutamic Acid as an Attachment Group.

In a still further interesting embodiment of the invention, thenon-polypeptide moiety has aspartic acid or glutamic acid as anattachment group, i.e., the conjugate of the invention is one whichcomprises at least one non-polypeptide moiety covalently attached to apolypeptide, wherein the amino acid sequence of the polypeptide differsfrom that of wild-type FVII or FVIIa shown in SEQ ID NO:1 in that atleast one aspartic acid residue and/or at least one glutamic acidresidue has been introduced or removed.

In a further interesting embodiment, the amino acid sequence of the FVIIor FVIIa polypeptide differs from that shown in SEQ ID NO:1 in that atleast one aspartic acid residue and/or glutamic acid residue, such as1-15 aspartic acid residues and/or glutamic acid residues, in particular1-10, 1-6 or 2-4 aspartic acid residues and/or glutamic acid residues,has been introduced, preferably by substitution.

Examples of positions, wherein aspartic acid residues or glutamic acidresidues can be introduced include, but is not limited to, positions ator in the vicinity of the proteolytic degradation sites described above.

Thus, in an interesting embodiment of the invention, the aspartic acidresidue and/or the glutamic acid residue to be introduced, preferably bysubstitution, is selected from the group consisting of I30D/E, K32D/E,A34D/E, T37D/E, K38D/E, W41D/E, Y44D/E, S45D/E, D46C, L141D/E, E142D/E,K143D/E, R144D/E, L288D/E, R290D/E, G291D/E, A292D/E, Q313D/E, S314D/E,R315D/E, K316D/E, V317D/E, L390D/E, M391D/E, R392D/E, S393D/E, P395D/E,R396D/E, P397D/E, G398D/E, V399D/E, L401D/E, R402D/E, A403D/E, P404D/E,and combinations thereof, in particular selected from the groupconsisting of K32D/E, Y44D/E, K143D/E, R290D/E, R315D/E, K341D/E,R392D/E, R396D/E, R402D/E and combinations thereof.

In addition to the above listed substitution(s), the polypeptide of theconjugate according to the above embodiment can comprise removal,preferably by substitution, of at least one of the aspartic acid residueand/or at least one glutamic acid residue.

Due to the relative high amount of lysine residues in the parentpolypeptide it is envisaged that at least one aspartic acid or glutamicacid residue should preferably be removed, in particular bysubstitution, in order to avoid excessive conjugation to non-polypeptidemoieties.

Thus, in one embodiment, the amino acid sequence of the FVII or FVIIapolypeptide part of the conjugate differs from that shown in SEQ ID NO:1in that at least one aspartic acid or glutamic acid residue, such as1-15 aspartic acid or glutamic acid residues, in particular 1-10, 1-6 or2-4 aspartic acid or glutamic acid residues, has been removed,preferably by substitution. For example, the aspartic acid and glutamicacid residue(s) to be removed, preferably by substitution, is selectedfrom the group consisting of D33, D46, D48, E77, E82, D86, D87, E94,E99, D104, E116, D123, E132, E142, E163, D196, E210, D212, E215, D217,D219, E220, D256, E265, E270, D289, E296, D309, D319, E325, D334, D338,D343, E385, E394 and combinations thereof.

It will be understood that it is particularly preferred that at leastone of the aspartic acid or glutamic acid residues, which are introducedat a predetermined site in the parent molecule, is combined with atleast one of the above-mentioned removals of aspartic acid or glutamicacid residues. Thus, in a preferred embodiment of the invention, theamino acid sequence of the FVII or FVIIa polypeptide part of theconjugate differs from that shown in SEQ ID NO:1 in that at least oneaspartic acid or glutamic acid residue has been removed, preferably bysubstitution, and at least one aspartic acid or glutamic acid residuehas been introduced, preferably by substitution.

While the non-polypeptide moiety of the conjugate according to thisaspect of the invention, which has an aspartic acid group or a glutamicacid group as an attachment group, can be any non-polypeptide moietywith such property, it is presently preferred that the non-polypeptidemoiety is a polymer molecule or an organic derivatizing agent, inparticular a polymer molecule, and the conjugate is prepared, e.g., asdescribed by Sakane and Pardridge, Pharmaceutical Research, Vol. 14, No.8, 1997, pp 1085-1091.

It will be understood that any of the amino acid changes, in particularsubstitutions, specified in this section can be combined with any of theamino acid changes, in particular substitutions specified in the othersections herein disclosing specific amino acid changes, includingintroduction and/or removal of glycosylation sites.

Conjugate of the Invention wherein the Non-Polypeptide Moiety is a SugarMoiety

In a further interesting embodiment of the invention, an attachmentgroup for a sugar moiety, such as a glycosylation site, in particular anin vivo glycosylation site, has been inserted and/or removed.

Preferably, the conjugate of the invention is one which comprises atleast one sugar moiety covalently attached to a polypeptide, wherein theamino acid sequence of the polypeptide differs from that of wild-typeFVII or FVIIa shown in SEQ ID NO:1 in that at least one non-naturallyoccurring glycosylation site has been introduced and/or at least onenaturally occurring glycosylation site has been removed. In particular,a non-naturally occurring glycosylation site has been introduced, or anon-naturally occurring glycosylation site has been introduced incombination with the removal of a natural occurring glycosylation site.The introduced glycosylation site can be an O-glycosylation site or anN-glycosylation site. Preferably the glycosylation site is an in vivoO-glycosylation site or an in vivo N-glycosylation site, in particularan in vivo N-glycosylation site.

When used in the present context, the term “naturally occurringglycosylation site” covers the glycosylation sites at postions N145,N322, S52 and S60. In a similar way, the term “naturally occurring invivo O-glycosylation site” includes the positions S52 and S60, whereasthe term “naturally occurring in vivo N-glycosylation site” includespositions N145 and N322.

Typically, the amino acid sequence of the FVII or FVIIa polypeptidediffers from that shown in SEQ ID NO:1 in that at least onenon-naturally occurring glycosylation site (e.g., at least onenon-naturally occurring in vivo N-glycosylation site), such as 1-15non-naturally occurring glycosylation sites (e.g., 1-15 non-naturallyoccurring in vivo N-glycosylation sites), in particular 1-10, 1-6 or 2-4non-naturally occurring glycosylation sites (e.g., 1-10, 1-6 or 2-4non-naturally occurring in vivo N-glycosylation sites), has beenintroduced, preferably by substitution.

It will be understood that in order to prepare a conjugate, wherein thepolypeptide of the conjugate comprises one or more glycosylation sites,the polypeptide must be expressed in a host cell capable of attachingsugar (oligosaccharide) moieties at the glycosylation site(s) oralternatively subjected to in vitro glycosylation. Examples ofglycosylating host cells are given in the section further below entitled“Coupling to a sugar moiety.”

In an interesting embodiment of this aspect, an in vivo glycosylationsite is introduced into a position of the parent FVII or FVIIa moleculeoccupied by an amino acid residue exposed to the surface of themolecule, preferably with more than 25% of the side chain exposed to thesolvent, in particular more than 50% exposed to the solvent (thesepositions are identified in the Methods section herein). TheN-glycosylation site is then introduced in such a way that the N-residueof said site is located in said position. Analogously, anO-glycosylation site is introduced so that the S or T residue making upsuch site is located in said position. Examples of such positionsinclude K32S/T, I42S/T, Y44S/T, K143S/T, R290S/T, R315S/T, K341S/T,R392S/T, R396S/T, R402S/T and combinations thereof.

With respect to N-glycosylation, the in vivo glycosylation site isintroduced into a position wherein only one mutation is required tocreate the site (i.e., where any other amino acid residues required forcreating a functional glycosylation site is already present in themolecule).

In other words, the conjugate according to the invention is preferably aconjugate, wherein the amino acid sequence of the polypeptide differsfrom SEQ ID NO:1 in that at least one naturally occurring N-X′-Xsequence is substituted with a N-X′-S or N-X′-T sequence, wherein X′ isany amino acid except P, and X is any amino acid except for S and T.

In a similar way, the conjugate according to the invention is preferablya conjugate, wherein the amino acid sequence of the polypeptide differsfrom SEQ ID NO:1 in that at least one naturally occurring X-X′-S orX-X′-T sequence naturally present in SEQ ID NO:1 is substituted with aN-X′-S or a N-X′-T sequence, wherein X′ is any amino acid except P, andX is an amino acid except for N.

Specific examples of such substitutions creating an in vivoN-glycosylation site include a substitution selected from the groupconsisting of F4S/T, P10ON, Q21N, W41N, S43N, A51N, G58N, L65N, G59S/T,E82S/T, N95S/T, G97S/T, Y101N, D104N, T106N, K109N, G117N, G124N, S126N,T128N, A175S/T, G179N, I186S/T, V188N, R202S/T, I205S/T, D212N, E220N,I230N, P231N, P236N, G237N, V253N, E265N, T267N, E270N, R277N, L280N,G291N, P303S/T, L305N, Q312N, G318N, G331N, D334N, K337N, G342N, H348N,R353N, Y357N, I361N, V376N, R379N, M391N, and combinations thereof.Preferably, the substitution is selected from the group consisting ofF4S/T, P10N, Q21N, W41N, A51N, G58N, G59S/T, N95S/T, G97S/T, Y11N,D104N, T106N, K109N, G117N, G124N, S126N, T128N, A175S/T, I186S/T,V188N, R202S/T, I205S/T, D212N, E220N, V253N, E265N, T267N, E270N,L280N, G291N, P303S/T, G318N, G331N, D334N, K337N, R353N, Y357N, M391N,and combinations thereof.

Alternatively, the in vivo glycosylation site is introduced in aposition occupied by a lysine residue or an arginine residue, preferablyoccupied by an lysine residue, in particular so that the N-residue ofthe N-glycosylation site or the S or T residue of an O-glycosylationsite substitutes the lysine residue.

Stated differently, the conjugate according to the invention ispreferably a conjugate, wherein the amino acid sequence of thepolypeptide differs from SEQ ID NO:1 in that at least one K-X′-Xsequence or R-X′-X sequence, preferably a K-X′-X sequence, naturallypresent in SEQ ID NO:1 is substituted with a N-X′-S or a N-X′-Tsequence, wherein X′ is any amino acid except P, and X is any amino acidexcept for S and T.

Specific examples of such substitutions creating an in vivoN-glycosylation site include a substitution selected from the groupconsisting of K32N+A34S/T, K38N+F40S/T, K62N+Q64S/T, K85N+D87S/T,K137N+P139S/T, K143N+N145S/T, K148N+Q149S/T, K161N+E163S/T,K197N+K199S/T, K199N+W201S/T, K316N+G318S/T, K337N, K341N+D343S/T,K389N+M391S/T and combinations thereof.

In a further interesting embodiment of the glycosylation aspectdescribed above, a glycosylation site, in particular an N-glycosylationsite, can be introduced in positions at or in the vicinity of theproteolytic degradation sites described above (see the section entitled“Conjugate of the invention”).

Thus, specific examples of such substitutions creating an in vivoN-glycosylation site include a substitution selected from the groupconsisting of K32N+A34S/T, F31N+D33S/T, I30N+K32S/T, A34N+R36S/T,K38N+F40S/T, T37N+L39S/T, R36N+K38S/T, L39N+W41S/T, F40N+I42S/T, W41N,I42N+Y44S/T, S43N, Y44N+D46S/T, S45N+G47S/T, D46N+D48S/T, G47N+Q49S/T,K143N+N145S/T, E142N+R144S/T, L141N+K143S/T, I140N+E142S/T,R144N+A146S/T, A146N+K148S/T, S147N+P149S/T, R290N+A292S/T,D289N+G291S/T, L288N+R290S/T, L287N+D289S/T, G291N, A292N+A294S/T,T293N+L295S/T, R315N+V317S/T, S314N+K316S/T, Q313N+R315S/T, Q312N,K316N+G318S/T, V317N+D319S/T, G318N, K341N+D343S/T, S339N+K341S/T,G342N, D343N+G345S/T, R392N+E394S/T, M391N, L390N+R392S/T,K389N+M391S/T, S393N+P395S/T, E394N+R396S/T, P395N+P397S/T,R396N+G398S/T, P397N+V399S/T, G398N+L400S/T, V399N+L401S/T,L400N+R402S/T, L401N+A403S/T, R402N+P404S/T, A403N+F405S/T,P404N+P406S/T and combinations thereof, such asK143N+N145S/T+R315N+V317S/T. Preferably, the substitution is selectedfrom the group consisting of K32N+A34S/T, K38N+F40S/T, Y44N+D46S/T,K143N+N145S/T, R290N+A292S/T, R315N+V317S/T, K341N+D343S/T,R392N+E394S/T, R396N+G398S/T, R402N+P404S/T and combinations thereof,such as K143N+N145S/T+R315N+V317S/T. More preferably, the substitutionis selected from the group consisting of K32N+A34T, K38N+F40T,Y44N+D46T, K143N+N145T, R290N+A292T, R315N+V317T, 341N+D343T,R392N+E394T, R396N+G398T, R402N+P404T and combinations thereof, inparticular K143N+N145T+R315N+V317T.

In a still further interesting embodiment of the invention, it ispreferred that the in vivo glycosylation site is introduced in aposition which does neither form part of the tissue factor binding norform part of the active site region and the ridge of the active sitebinding cleft as defined herein. It is envisaged that such glycosylationvariants will primarily belong to the class of active conjugates asdefined hereinbefore.

Thus, specific examples of substitutions creating such an in vivoN-glycosylation site include substitutions selcted from the groupconsisting of K32N+A34S/T, I30N+K32S/T, A34N+R36S/T, K38N+F40S/T,T37N+L39S/T, W41N, Y44N+D46S/T, S45N+G47S/T, D46N+D48S/T, G47N+Q49S/T,K143N+N145S/T, E142N+R144S/T, L141N+K143S/T, I140N+E142S/T,R144N+A146S/T, A146N+K148S/T, S147N+P149S/T, L288N+R290S/T,L287N+D289S/T, R315N+V317S/T, S314N+K316S/T, K316N+G318S/T,V317N+D319S/T, G318N, R392N+E394S/T, M391N, L390N+R392S/T,K389N+M391S/T, S393N+P395S/T, E394N+R396S/T, P395N+P397S/T,R396N+G398S/T, P397N+V399S/T, G398N+L400S/T, V399N+L401S/T,L401N+A403S/T, R402N+P404S/T, A403N+F405S/T, P404N+P406S/T andcombinations thereof, such as K143N+N145S/T+R315N+V317S/T. Preferably,the substitution is selcted from the group consisting of K32N+A34S/T,K38N+F40S/T, Y44N+D46S/T, K143N+N145S/T, R315N+V317S/T, R392N+E394S/T,R396N+G398S/T, R402N+P404S/T and combinations thereof, such asK143N+N145S/T+R315N+V317S/T. More preferably, the substitution isselected from the group consisting of K32N+A34T, K38N+F40T, Y44N+D46T,K143N+N145T, R315N+V317T, R392N+E394T, R396N+G398T, R402N+P404T andcombinations thereof, in particular K143N+N145T+R315N+V317T.

In an even further interesting embodiment of the invention, it ispreferred that the in vivo glycosylation site is introduced in aposition which does not form part of the tissue factor but which formspart of the active site region and the ridge of the active site bindingcleft as defined herein. It is envisaged that such glycosylationvariants will primarily belong to the class of inactive conjugates asdefined hereinbefore.

Thus, specific examples of substitutions creating such an in vivoN-glycosylation site include substitutions selcted from the groupconsisting of I153N+G155S/T, Q167N+L169S/T, V168N+L170S/T,L169N+L171S/T, L170N+V172S/T, L171N+N173S/T, A175S/T, A175N+L177S/T,L177N+G179S/T, G179N, G180N+L182S/T, T181N+I183S/T, V188N,V189N+A191S/T, S190N+A192S/T, A191N+H193S/T, H193N+F195S/T,F195N+K197S/T, D196N+I198S/T, K197N+K199S/T, I198N+N200S/T,K199N+W201S/T, W201N+N203S/T, R202S/T, I205S/T, V228N+I230S/T,I229N+P231S/T, I230N, P231N, S232N+Y234S/T, T233N+V235S/T,Y234N+P236S/T, V235N+G237S/T, P236N, G237N, T238N+N240S/T,T239N+H241S/T, H241N+I243S/T, D242S/T, I243N+L245S/T, A244N+L246S/T,L245N+R247S/T, L246N+L246S/T, V281N+G283S/T, S282N+W284S/T,G283N+G285S/T, W284N+Q286S/T, G285N+L287S/T, Q286N+L288S/T,D289N+G291S/T, R290N+A292S/T, G291N, A292N+A294S/T, T293N+L295S/T,P321N+I323S/T, T324N+Y326S/T, E325N+M327S/T, Y326N+F327S/T,F328N+A330S/T, S339N+K341S/T, K341N+D343S/T, G342N+S344S/T,D343N+G345S/T, S344N+G346S/T, G345N+P347S/T, P347N+A349S/T, H348N,L358N+G360S/T, T359N+I361S/T, G360N+V362S/T, I361N, V362N+W364S/T,S363N+G365S/T, W364N+Q366S/T, G365N+G367S/T, T370N+G372S/T, V376N,Y377N+R379S/T, T378N+V380S/T, R379N, V380N+Q382S/T, Q382N+I384S/T,Y383N+E385S/T, W386N+Q388S/T, L387N+K389S/T, L400N+R402S/T andcombinations thereof. Preferably, the substitution is selected from thegroup consisting of D289N+G291S/T, R290N+A292S/T, G291N, A292N+A294S/T,T293N+L295S/T, S339N+K341S/T, K341N+D343S/T, G342N+S344S/T,D343N+G345S/T, and combinations thereof. More preferably, thesubstitution is selcted from the group consisting of D289N+G291T,R290N+A292T, G291N, A292N+A294T, T293N+L295T, S339N+K341T, K341N+D343T,G342N+S344T, D343N+G345T, and combinations thereof.

In addition to a sugar moiety, the conjugate according to the aspect ofthe invention described in the present section can contain additionalnon-polypeptide moieties, in particular a polymer molecule, as describedin the present application, conjugated to one or more attachment groupspresent in the polypeptide part of the conjugate.

It will be understood that any of the amino acid changes, in particularsubstitutions, specified in this section can be combined with any of theamino acid changes, in particular substitutions, specified in the othersections herein disclosing specific amino acid changes.

For instance, any of the glycosylated variants disclosed in the presentsection having introduced and/or removed at least one glycosylationsite, such as a variant comprising the substitutions R315N+V317T and/orK143N+N145T, can further be conjugated to a polymer molecule, such asPEG, or any other non-polypeptide moiety. For this purpose theconjugation can be achieved by use of attachment groups already presentin the FVII or FVIIa polypeptide or attachment groups can have beenintroduced and/or removed, in particular such that a total of 1-6, inparticular 3-4 or 1, 2, 3, 4, 5, or 6 attachment groups are availablefor conjugation.

Preferably, in a conjugate of the invention wherein the FVII or FVIIapolypeptide comprises two glycosylation sites, the number and molecularweight of the non-polypeptide moiety is chosen so as that the totalmolecular weight added by the non-polypeptide moiety is in the range of5-25 kDa, such as in the range of 10-25 kDa, in particular about 5 kDa,about 12 kDa, about 15 kDa or about 20 kDa.

An Inactive Conjugate

The conjugates of the invention can be rendered inactive by removing atleast one amino acid residue occupying a position selected from thegroup consisting of R152, I153, S344, D242 and H193 of SEQ ID NO:1. Theremoval can be effected by substitution or deletion of one or more ofthe above-identified amino acid residues. Preferably, the removal iseffected by substitution, in particular by conservative substitution.Accordingly, the inactive FVII or FVII a polypeptide used herein cancomprise one or more of the following substitutions: R152X, I153X,S344X, D242X or H193X, wherein X is any amino acid residue, preferablyone leading to a conservative substitution. For instance, the inactiveFVII or FVIIa polypeptide comprises the mutations R152X, wherein X isany amino acid residue other than lysine (since lysine forms part of aprotease cleavage site). Other examples of specific substitutionsinclude I153A/V/L; S344T/A/G/Y; D242E/A and/or H193R/A.

Alternatively, an active FVII or FVIIa polypeptide can be renderedinactive by carbamylating the α-amino acid group I153 or by complexingthe polypeptide to a serine proteinase inhibitor. A suitable serineinhibitor protein is, e.g., selected from the group consisting of anorganophosphor coumpound, a sulfanylfluoride, a peptidehalomethylketone, preferably a Dansyl-Phe-Pro-Arg chloromethylketone,Dansyl-Glu-Glu-Arg chlormethylketone, Dansyl-Phe-Phe-Argchlormethylketone or a Phe-Phe-Arg chlormethylketone, or an azapeptide.

A conjugate can also be rendered inactive by introducing at least oneglycosylation site in a position selected so that the subsequentglycosylation inactivates the conjugate.

As explained above in the last part of the section entitled “Conjugateof the invention wherein the non-polypeptide moiety is a sugar moiety”it is preferred that such glycosylation sites are introduced in aposition which does not form part of the tissue factor but which formspart of the active site region and the ridge of the active site bindingcleft as defined herein. Specific examples of preferred substitutionsare given above in the section entitled “Conjugate of the inventionwherein the non-polypeptide moiety is a sugar moiety”.

Non-Polypeptide Moiety of the Conjugate of the Invention

As indicated further above the non-polypeptide moiety of the conjugateof the invention is preferably selected from the group consisting of apolymer molecule, a lipophilic compound, a sugar moiety (by way of invivo glycosylation) and an organic derivatizing agent. All of theseagents can confer desirable properties to the polypeptide part of theconjugate, in particular increased functional in vivo half-life and/orincreased plasma half-life. The polypeptide part of the conjugate isnormally conjugated to only one type of non-polypeptide moiety, but canalso be conjugated to two or more different types of non-polypeptidemoieties, e.g., to a polymer molecule and a sugar moiety, to alipophilic group and a sugar moiety, to an organic derivatizing agentand a sugar moiety, to a lipophilic group and a polymer molecule, etc.The conjugation to two or more different non-polypeptide moieties can bedone simultaneous or sequentially.

Methods of Preparing a Conjugate of the Invention

In the following sections “Conjugation to a lipophilic compound,”“Conjugation to a polymer molecule,” “Conjugation to a sugar moiety” and“Conjugation to an organic derivatizing agent” conjugation to specifictypes of non-polypeptide moieties is described. In general, apolypeptide conjugate according to the invention can be produced byculturing an appropriate host cell under conditions conducive for theexpression of the polypeptide, and recovering the polypeptide, whereina) the polypeptide comprises at least one N- or O-glycosylation site andthe host cell is a eukaryotic host cell capable of in vivoglycosylation, and/or b) the polypeptide is subjected to conjugation toa non-polypeptide moiety in vitro.

It will be understood that the conjugation should be designed so as toproduce the optimal molecule with respect to the number ofnon-polypeptide moieties attached, the size and form of such molecules(e.g., whether they are linear or branched), and the attachment site(s)in the polypeptide. The molecular weight of the non-polypeptide moietyto be used can e.g., be chosen on the basis of the desired effect to beachieved. For instance, if the primary purpose of the conjugation is toachieve a conjugate having a high molecular weight (e.g., to reducerenal clearance) it is usually desirable to conjugate as few highmolecular weight non-polypeptide moieties as possible to obtain thedesired molecular weight. When a high degree of shielding is desirablethis can be obtained by use of a sufficiently high number of lowmolecular weight non-polypeptide moieties (e.g., with a molecular weightof from about 300 Da to about 5 kDa, such as a molecular weight of from300 Da to 2 kDa) to effectively shield all or most protease cleavagesites or other vulnerable sites of the polypeptide.

Conjugation to a Lipophilic Compound

The polypeptide and the lipophilic compound can be conjugated to eachother, either directly or by use of a linker. The lipophilic compoundcan be a natural compound such as a saturated or unsaturated fatty acid,a fatty acid diketone, a terpene, a prostaglandin, a vitamine, acarotenoide or steroide, or a synthetic compound such as a carbon acid,an alcohol, an amine and sulphonic acid with one or more alkyl-, aryl-,alkenyl- or other multiple unsaturated compounds. The conjugationbetween the polypeptide and the lipophilic compound, optionally througha linker can be done according to methods known in the art, e.g., asdescribed by Bodanszky in Peptide Synthesis, John Wiley, New York, 1976and in WO 96/12505.

Conjugation to a Polymer Molecule, Including Conjugation of a PolymerMolecule to the N-Terminal of the Polypeptide

The polymer molecule to be coupled to the polypeptide can be anysuitable polymer molecule, such as a natural or synthetic homo-polymeror hetero-polymer, typically with a molecular weight in the range ofabout 300-100,000 Da, such as about 500-20,000 Da, more preferably inthe range of about 500-15,000 Da, even more preferably in the range ofabout 2-12 kDa, such as in the range of about 3-10 kDa. When the term“about” is used herein in connection with a certain molecular weight,the word “about” indicates an approximate average molecular weight andreflects the fact that there will normally be a certain molecular weightdistribution in a given polymer preparation.

Examples of homo-polymers include a polyol (i.e., poly-OH), a polyamine(i.e., poly-NH₂) and a polycarboxylic acid (i.e., poly-COOH). Ahetero-polymer is a polymer comprising different coupling groups, suchas a hydroxyl group and an amine group.

Examples of suitable polymer molecules include polymer moleculesselected from the group consisting of polyalkylene oxide (PAO),including polyalkylene glycol (PAG), such as polyethylene glycol (PEG)and polypropylene glycol (PPG), branched PEGs, poly-vinyl alcohol (PVA),poly-carboxylate, poly-(vinylpyrolidone), polyethylene-co-maleic acidanhydride, polystyrene-co-maleic acid anhydride, dextran, includingcarboxymethyl-dextran, or any other biopolymer suitable for reducingimmunogenicity and/or increasing functional in vivo half-life and/orserum half-life. Another example of a polymer molecule is human albuminor another abundant plasma protein. Generally, polyalkyleneglycol-derived polymers are biocompatible, non-toxic, non-antigenic,non-immunogenic, have various water solubility properties, and areeasily excreted from living organisms.

PEG is the preferred polymer molecule, since it has only few reactivegroups capable of cross-linking compared to, e.g., polysaccharides suchas dextran. In particular, mono-functional PEG, e.g.,methoxypolyethylene glycol (mPEG), is of interest since its couplingchemistry is relatively simple (only one reactive group is available forconjugating with attachment groups on the polypeptide). Consequently,the risk of cross-linking is eliminated, the resulting polypeptideconjugates are more homogeneous and the reaction of the polymermolecules with the polypeptide is easier to control.

To effect covalent attachment of the polymer molecule(s) to thepolypeptide, the hydroxyl end groups of the polymer molecule must beprovided in activated form, i.e., with reactive functional groups(examples of which include primary amino groups, hydrazide (HZ), thiol,succinate (SUC), succinimidyl succinate (SS), succinimidyl succinamide(SSA), succinimidyl proprionate (SPA), succinimidy carboxymethylate(SCM), benzotriazole carbonate (BTC), N-hydroxysuccinimide (NHS),aldehyde, nitrophenylcarbonate (NPC), and tresylate (TRES)). Suitableactivated polymer molecules are commercially available, e.g., fromShearwater Polymers, Inc., Huntsville, Ala., USA, or from PolyMASCPharmaceuticals plc, UK.

Alternatively, the polymer molecules can be activated by conventionalmethods known in the art, e.g., as disclosed in WO 90/13540. Specificexamples of activated linear or branched polymer molecules for use inthe present invention are described in the Shearwater Polymers, Inc.1997 and 2000 Catalogs (Functionalized Biocompatible Polymers forResearch and pharmaceuticals, Polyethylene Glycol and Derivatives,incorporated herein by reference).

Specific examples of activated PEG polymers include the following linearPEGs: NHS-PEG (e.g., SPA-PEG, SSPA-PEG, SBA-PEG, SS-PEG, SSA-PEG,SC-PEG, SG-PEG, and SCM-PEG), and NOR-PEG, BTC-PEG, EPOX-PEG, NCO-PEG,NPC-PEG, CDI-PEG, ALD-PEG, TRES-PEG, VS-PEG, IODO-PEG, and MAL-PEG, andbranched PEGs such as PEG2-NHS and those disclosed in U.S. Pat. No.5,932,462 and U.S. Pat. No. 5,643,575, both of which are incorporatedherein by reference. Furthermore, the following publications,incorporated herein by reference, disclose useful polymer moleculesand/or PEGylation chemistries: U.S. Pat. No. 5,824,778, U.S. Pat. No.5,476,653, WO 97/32607, EP 229,108, EP 402,378, U.S. Pat. No. 4,902,502,U.S. Pat. No. 5,281,698, U.S. Pat. No. 5,122,614, U.S. Pat. No.5,219,564, WO 92/16555, WO 94/04193, WO 94/14758, WO 94/17039, WO94/18247, WO 94/28024, WO 95/00162, WO 95/11924, WO95/13090, WO95/33490, WO 96/00080, WO 97/18832, WO 98/41562, WO 98/48837, WO99/32134, WO 99/32139, WO 99/32140, WO 96/40791, WO 98/32466, WO95/06058, EP 439 508, WO 97/03106, WO 96/21469, WO 95/13312, EP 921 131,U.S. Pat. No. 5,736,625, WO 98/05363, EP 809 996, U.S. Pat. No.5,629,384, WO 96/41813, WO 96/07670, U.S. Pat. No. 5,473,034, U.S. Pat.No. 5,516,673, EP 605 963, U.S. Pat. No. 5,382,657, EP 510 356, EP 400472, EP 183 503 and EP 154 316.

The conjugation of the polypeptide and the activated polymer moleculesis conducted by use of any conventional method, e.g., as described inthe following references (which also describe suitable methods foractivation of polymer molecules): R. F. Taylor, (1991), “Proteinimmobilisation. Fundamental and applications,” Marcel Dekker, N.Y.; S.S. Wong, (1992), “Chemistry of Protein Conjugation and Crosslinking,”CRC Press, Florida, USA; G. T. Hermanson et al., (1993), “ImmobilizedAffinity Ligand Techniques,” Academic Press, N.Y.). The skilled personwill be aware that the activation method and/or conjugation chemistry tobe used depends on the attachment group(s) of the polypeptide (examplesof which are given further above), as well as the functional groups ofthe polymer (e.g., being amine, hydroxyl, carboxyl, aldehyde, sulfydryl,succinimidyl, maleimide, vinysulfone or haloacetate). The PEGylation canbe directed towards conjugation to all available attachment groups onthe polypeptide (i.e., such attachment groups that are exposed at thesurface of the polypeptide) or can be directed towards one or morespecific attachment groups, e.g., the N-terminal amino group asdescribed in U.S. Pat. No. 5,985,265. Furthermore, the conjugation canbe achieved in one step or in a stepwise manner (e.g., as described inWO 99/55377).

It will be understood that the PEGylation is designed so as to producethe optimal molecule with respect to the number of PEG moleculesattached, the size and form of such molecules (e.g., whether they arelinear or branched), and the attachment site(s) in the polypeptide. Themolecular weight of the polymer to be used can e.g., be chosen on thebasis of the desired effect to be achieved. For instance, if the primarypurpose of the conjugation is to achieve a conjugate having a highmolecular weight (e.g., to reduce renal clearance) it is usuallydesirable to conjugate as few high molecular weight polymer molecules aspossible to obtain the desired molecular weight. When a high degree ofshielding is desirable this can be obtained by use of a sufficientlyhigh number of low molecular weight polymer molecules (e.g., with amolecular weight of from about 300 Da to about 5 kDa) to effectivelyshield all or most protease cleavage sites or other vulnerable sites ofthe polypeptide. For instance, 2-8, such as 3-6 such polymers can beused.

In connection with conjugation to only a single attachment group on theprotein (e.g., the N-terminal amino group), it can be advantageous thatthe polymer molecule, which can be linear or branched, has a highmolecular weight, preferably about 10-25 kDa, such as about 15-25 kDa,e.g., about 20 kDa.

Normally, the polymer conjugation is performed under conditions aimed atreacting as many of the available polymer attachment groups with polymermolecules. This is achieved by means of a suitable molar excess of thepolymer relative to the polypeptide. Typically, the molar ratios ofactivated polymer molecules to polypeptide are up to about 1000-1, suchas up to about 200-1, or up to about 100-1. In some cases the ration canbe somewhat lower, however, such as up to about 50-1, 10-1 or 5-1 inorder to obtain optimal reaction.

It is also contemplated according to the invention to couple the polymermolecules to the polypeptide through a linker. Suitable linkers are wellknown to the skilled person. A preferred example is cyanuric chloride(Abuchowski et al., (1977), J. Biol. Chem., 252, 3578-3581; U.S. Pat.No. 4,179,337; Shafer et al., (1986), J. Polym. Sci. Polym. Chem. Ed.,24, 375-378).

Subsequent to the conjugation, residual activated polymer molecules areblocked according to methods known in the art, e.g., by addition ofprimary amine to the reaction mixture, and the resulting inactivatedpolymer molecules are removed by a suitable method.

It will be understood that depending on the circumstances, e.g., theamino acid sequence of the polypeptide, the nature of the activated PEGcompound being used and the specific PEGylation conditions, includingthe molar ratio of PEG to polypeptide, varying degrees of PEGylation canbe obtained, with a higher degree of PEGylation generally being obtainedwith a higher ratio of PEG to polypeptide. The PEGylated polypeptidesresulting from any given PEGylation process will, however, normallycomprise a stochastic distribution of polypeptide conjugates havingslightly different degrees of PEGylation.

In an interesting embodiment of the invention, the polypeptide conjugateof the invention comprises a polymer molecule covalently attached to theA1 N-terminal of the wild type FVII or FVIIa polypeptide shown in SEQ IDNO:1, where said polymer molecule is the only polymer molecule attachedto the polypeptide. Preferably, such polypeptide conjugates are ones,which comprise a single PEG molecule attached to the N-terminal of thepolypeptide and no other PEG molecules. In particular, a linear orbranched PEG molecule with a molecular weight of at least about 5 kDa,in particular about 10-25 kDa, such as about 15-25 kDa, e.g., about 20kDa is preferred. The polypeptide conjugate according to this embodimentcan further comprise one or more sugar moieties attached to an N-linkedor O-linked glycosylation site of the polypeptide or sugar moietiesattached by in vitro glycosylation.

In a further interesting embodiment of the invention the polypeptideconjugate of the invention comprises polymer molecules covalentlyattached to the A1 N-terminal and to the I153 N-terminal of the wildtype FVIIa polypeptide shown in SEQ ID NO:1, where said polymermolecules are the only polymer molecules attached to the polypeptide.Preferably, such polypeptide conjugates are ones, which comprise a PEGmolecule attached to both of the N-terminals of FVIIa and no other PEGmolecules. In particular, linear or branched PEG molecules with amolecular weight of at least about 5 kDa, in particular about 10-25 kDa,such as about 15-25 kDa, e.g., about 20 kDa are preferred. Thepolypeptide conjugate according to this embodiment can further compriseone or more sugar moieties attached to an N-linked or O-linkedglycosylation site of the polypeptide or sugar moieties attached by invitro glycosylation.

One preferred method for selectively coupling polymer molecules, such asPEG molecules, to the N-terminal of the polypeptide is the methoddisclosed in U.S. Pat. No. 5,985,265. This method involves reductivealkylation (reaction of the N-terminal amino group of the polypeptidewith an aldehyde-containing polypeptide, such as aldehylde-PEG, in thepresence of a reducing agent, such as NaCNBH₃). This method exploitsdifferential reactivity of different types of primary amino groups(lysine versus N-terminal) available for derivatization in thepolypeptide, thereby achieving substantially selective derivatization ofthe polypeptide at the N-terminus with a carbonyl group-containingpolymer molecule, such as aldehyde-PEG. The reaction is performed at apH which allows one to take advantage of the pK_(a) differences betweenthe ε-amino groups of the lysine residues and that of the α-amino groupof the N-terminal residue of the polypeptide. In order to achieve thisdifferential reactivity, the reaction is typically carried at slightlyacidic conditions. Specific examples of suitable pH ranges include pH4.5-7, such as pH 4.5-6, e.g., pH 5-6, in particular about pH 5.

In another specific embodiment, the polypeptide conjugate of theinvention comprises a PEG molecule attached to each of the lysineresidues in the polypeptide available for PEGylation, in particular alinear or branched PEG molecule, e.g., with a molecular weight of about1-15 kDa, typically about 2-12 kDa, such as about 3-10 kDa, e.g., about5 or 6 kDa.

In yet another embodiment, the polypeptide conjugate of the inventioncomprises a PEG molecule attached to each of the lysine residues in thepolypeptide available for PEGylation, and in addition to the N-terminalamino acid residue of the polypeptide.

Covalent in vitro coupling of carbohydrate moieties (such as dextran) toamino acid residues of the polypeptide can also be used, e.g., asdescribed, for example in WO 87/05330 and in Aplin etl al., CRC CritRev. Biochem, pp. 259-306, 1981. The in vitro coupling of carbohydratemoieties or PEG to protein- and peptide-bound Gln-residues can becarried out by transglutaminases (TGases). Transglutaminases catalysethe transfer of donor amine-groups to protein- and peptide-boundGln-residues in a so-called cross-linking reaction. The donor-aminegroups can be protein- or peptide-bound, such as the ε-amino-group inLys-residues or it can be part of a small or large organic molecule. Anexample of a small organic molecule functioning as amino-donor inTGase-catalysed cross-linking is putrescine (1,4-diaminobutane). Anexample of a larger organic molecule functioning as amino-donor inTGase-catalysed cross-linking is an amine-containing PEG (Sato et al.,1996, Biochemistry 35, 13072-13080).

TGases, in general, are highly specific enzymes, and not everyGln-residues exposed on the surface of a protein is accessible toTGase-catalysed cross-linking to amino-containing substances. On thecontrary, only few Gln-residues are naturally functioning as TGasesubstrates but the exact parameters governing which Gln-residues aregood TGase substrates remain unknown. Thus, in order to render a proteinsusceptible to TGase-catalysed cross-linking reactions it is often aprerequisite at convenient positions to add stretches of amino acidsequence known to function very well as TGase substrates. Several aminoacid sequences are known to be or to contain excellent natural TGasesubstrates e.g., substance P, elafin, fibrinogen, fibronectin,α₂-plasmin inhibitor, α-caseins, and β-caseins.

Coupling to a Sugar Moiety

In order to achieve in vivo glycosylation of a FVII molecule comprisingone or more glycosylation sites the nucleotide sequence encoding thepolypeptide must be inserted in a glycosylating, eucaryotic expressionhost. The expression host cell can be selected from fungal (filamentousfungal or yeast), insect or animal cells or from transgenic plant cells.In one embodiment, the host cell is a mammalian cell, such as a CHOcell, BHK or HEK, e.g., HEK 293, cell, or an insect cell, such as an SF9cell, or a yeast cell, e.g., S. cerevisiae or Pichia pastoris, or any ofthe host cells mentioned hereinafter.

Coupling to an Organic Derivatizing Agent

Covalent modification of the polypeptide can be performed by reactingone or more attachment groups of the polypeptide with an organicderivatizing agent. Suitable derivatizing agents and methods are wellknown in the art. For example, cysteinyl residues most commonly arereacted with α-haloacetates (and corresponding amines), such aschloroacetic acid or chloroacetamide, to give carboxymethyl orcarboxyamidomethyl derivatives. Cysteinyl residues also are derivatizedby reaction with bromotrifluoroacetone, α-bromo-β-(4-imidozoyl)propionicacid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyldisulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate,2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.Histidyl residues are derivatized by reaction withdiethylpyrocarbonateat pH 5.5-7.0 because this agent is relativelyspecific for the histidyl side chain. Para-bromophenacyl bromide also isuseful. The reaction is preferably performed in 0.1 M sodium cacodylateat pH 6.0. Lysinyl and amino terminal residues are reacted with succinicor other carboxylic acid anhydrides. Derivatization with these agentshas the effect of reversing the charge of the lysinyl residues. Othersuitable reagents for derivatizing α-amino-containing residues includeimidoesters such as methyl picolinimidate, pyridoxal phosphate,pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid,O-methylisourea, 2,4-pentanedione and transaminase-catalyzed reactionwith glyoxylate. Arginyl residues are modified by reaction with one orseveral conventional reagents, among them phenylglyoxal,2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization ofarginine residues requires that the reaction be performed in alkalineconditions because of the high pKa of the guanidine functional group.

Furthermore, these reagents can react with the groups of lysine as wellas the arginine guanidino group. Carboxyl side groups (aspartyl orglutamyl) are selectively modified by reaction with carbodiimides(R—N═C═N—R′), where R and R′ are different alkyl groups, such as1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore,aspartyl and glutamyl residues are converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Blocking of Functional Site

It has been reported that excessive polymer conjugation can lead to aloss of activity of the polypeptide to which the non-polypeptide moietyis conjugated. This problem can be eliminated, e.g., by removal ofattachment groups located at the functional site or by reversibleblocking the functional site prior to conjugation so that the functionalsite is blocked during conjugation. The latter strategy constitutesfurther embodiments of the invention (the first strategy beingexemplified further above, e.g., by removal of lysine residues which canbe located close to the functional site). More specifically, accordingto the second strategy the conjugation between the polypeptide and thenon-polypeptide moiety is conducted under conditions where thefunctional site of the polypeptide is blocked by a helper molecule e.g.,tissue factor capable of binding to the functional site of thepolypeptide or a serine protease inhibitor.

Preferably, the helper molecule is one, which specifically recognizes afunctional site of the polypeptide, such as a receptor, in particulartissue factor, either full length or a suitably truncated form of tissuefactor or two molecules, one being tissue factor the other one being apeptide or peptide inhibitor binding to and thus protecting the areaaround the catalytic triad (preferably defined as amino acid residueswithin 10 Å of any atom in the catalytic triad).

Alternatively, the helper molecule can be an antibody, in particular amonoclonal antibody recognizing the FVII polypeptide. In particular, thehelper molecule can be a neutralizing monoclonal antibody.

The polypeptide is allowed to interact with the helper molecule beforeeffecting conjugation. This ensures that the functional site of thepolypeptide is shielded or protected and consequently unavailable forderivatization by the non-polypeptide moiety such, as a polymer.Following its elution from the helper molecule, the conjugate betweenthe non-polypeptide moiety and the polypeptide can be recovered with atleast a partially preserved functional site.

The subsequent conjugation of the polypeptide having a blockedfunctional site to a polymer, a lipophilic compound, a sugar moiety, anorganic derivatizing agent or any other compound is conducted in thenormal way, e.g., as described in the sections above entitled“Conjugation to . . . ”.

Irrespectively of the nature of the helper molecule to be used to shieldthe functional site of the polypeptide from conjugation, it is desirablethat the helper molecule is free from or comprises only few attachmentgroups for the non-polypeptide moiety of choice in part(s) of themolecule, where the conjugation to such groups will hamper thedesorption of the conjugated polypeptide from the helper molecule.Hereby, selective conjugation to attachment groups present innon-shielded parts of the polypeptide can be obtained and it is possibleto reuse the helper molecule for repeated cycles of conjugation. Forinstance, if the non-polypeptide moiety is a polymer molecule such asPEG, which has the epsilon amino group of a lysine or N-terminal aminoacid residue as an attachment group, it is desirable that the helpermolecule is substantially free from conjugatable epsilon amino groups,preferably free from any epsilon amino groups. Accordingly, in apreferred embodiment, the helper molecule is a protein or peptidecapable of binding to the functional site of the polypeptide, whichprotein or peptide is free from any conjugatable attachment groups forthe non-polypeptide moiety of choice.

In a further embodiment, the helper molecule is first covalently linkedto a solid phase such as column packing materials, for instance Sephadexor agarose beads, or a surface, e.g., reaction vessel. Subsequently, thepolypeptide is loaded onto the column material carrying the helpermolecule and conjugation carried out according to methods known in theart, e.g., as described in the sections above entitled “Conjugation to .. . .” This procedure allows the polypeptide conjugate to be separatedfrom the helper molecule by elution. The polypeptide conjugate is elutedby conventional techniques under physico-chemical conditions that do notlead to a substantive degradation of the polypeptide conjugate. Thefluid phase containing the polypeptide conjugate is separated from thesolid phase to which the helper molecule remains covalently linked. Theseparation can be achieved in other ways: For instance, the helpermolecule can be derivatised with a second molecule (e.g., biotin) thatcan be recognized by a specific binder (e.g., streptavidin). Thespecific binder can be linked to a solid phase thereby allowing theseparation of the polypeptide conjugate from the helper molecule-secondmolecule complex through passage over a second helper-solid phase columnwhich will retain, upon subsequent elution, the helper molecule-secondmolecule complex, but not the polypeptide conjugate. The polypeptideconjugate can be released from the helper molecule in any appropriatefashion. Deprotection can be achieved by providing conditions in whichthe helper molecule dissociates from the functional site of the FVII towhich it is bound. For instance, a complex between an antibody to whicha polymer is conjugated and an anti-idiotypic antibody can bedissociated by adjusting the pH to an acid or alkaline pH. Even morepreferred is the use of a conformation specific antibody that recognizesa Ca²⁺ specific conformation of FVII and consequently can be eluted withEDTA under mild conditions.

Attachment of Serine Protease Inhibitor

Attachment of a serine protease inhibitor can be performed in accordancewith the method described in WO 96/12800.

Conjugation of a Tagged Polypeptide

In an alternative embodiment, the polypeptide is expressed as a fusionprotein with a tag, i.e., an amino acid sequence or peptide stretch madeup of typically 1-30, such as 1-20 amino acid residues. Besides allowingfor fast and easy purification, the tag is a convenient tool forachieving conjugation between the tagged polypeptide and thenon-polypeptide moiety. In particular, the tag can be used for achievingconjugation in microtiter plates or other carriers, such as paramagneticbeads, to which the tagged polypeptide can be immobilised via the tag.The conjugation to the tagged polypeptide in, e.g., microtiter plateshas the advantage that the tagged polypeptide can be immobilised in themicrotiter plates directly from the culture broth (in principle withoutany purification) and subjected to conjugation. Thereby, the totalnumber of process steps (from expression to conjugation) can be reduced.Furthermore, the tag can function as a spacer molecule, ensuring animproved accessibility to the immobilised polypeptide to be conjugated.The conjugation using a tagged polypeptide can be to any of thenon-polypeptide moieties disclosed herein, e.g., to a polymer moleculesuch as PEG.

The identity of the specific tag to be used is not critical as long asthe tag is capable of being expressed with the polypeptide and iscapable of being immobilised on a suitable surface or carrier material.A number of suitable tags are commercially available, e.g., from UnizymeLaboratories, Denmark. For instance, the tag can consist of any of thefollowing sequences:

-   His-His-His-His-His-His-   Met-Lys-His-His-His-His-His-His-   Met-Lys-His-His-Ala-His-His-Gln-His-His-   Met-Lys-His-Gln-His-Gln-His-Gln-His-Gln-His-Gln-His-Gln-   Met-Lys-His-Gln-His-Gln-His-Gln-His-Gln-His-Gln-His-Gln-Gln    or any of the following:-   EQKLI SEEDL (a C-terminal tag described in Mol. Cell. Biol.    5:3610-16, 1985)-   DYKDDDDK (a C- or N-terminal tag)-   YPYDVPDYA

Antibodies against the above tags are commercially available, e.g., fromADI, Aves Lab and Research Diagnostics.

The subsequent cleavage of the tag from the polypeptide can be achievedby use of commercially available enzymes.

Methods of Preparing a Polypeptide of the Invention or the Polypeptideof the Conjugate of the Invention

The polypeptide of the present invention or the polypeptide part of aconjugate of the invention, optionally in glycosylated form, can beproduced by any suitable method known in the art. Such methods includeconstructing a nucleotide sequence encoding the polypeptide andexpressing the sequence in a suitable transformed or transfected host.Preferably, the host cell is a gammacarboxylating host cell such as amammalian cell. However, polypeptides of the invention can be produced,albeit less efficiently, by chemical synthesis or a combination ofchemical synthesis or a combination of chemical synthesis andrecombinant DNA technology.

A nucleotide sequence encoding a polypeptide or the polypeptide part ofa conjugate of the invention can be constructed by isolating orsynthesizing a nucleotide sequence encoding the parent FVII, such ashFVII with the amino acid sequence shown in SEQ ID NO:1 and thenchanging the nucleotide sequence so as to effect introduction (i.e.,insertion or substitution) or removal (i.e., deletion or substitution)of the relevant amino acid residue(s).

The nucleotide sequence is conveniently modified by site-directedmutagenesis in accordance with conventional methods. Alternatively, thenucleotide sequence is prepared by chemical synthesis, e.g., by using anoligonucleotide synthesizer, wherein oligonucleotides are designed basedon the amino acid sequence of the desired polypeptide, and preferablyselecting those codons that are favored in the host cell in which therecombinant polypeptide will be produced. For example, several smalloligonucleotides coding for portions of the desired polypeptide can besynthesized and assembled by PCR, ligation or ligation chain reaction(LCR) (Barany, PNAS 88:189-193, 1991). The individual oligonucleotidestypically contain 5′ or 3′ overhangs for complementary assembly.

Alternative nucleotide sequence modification methods are available forproducing polypeptide variants for high throughput screening, forinstance methods which involve homologous cross-over such as disclosedin U.S. Pat. No. 5,093,257, and methods which involve gene shuffling,i.e., recombination between two or more homologous nucleotide sequencesresulting in new nucleotide sequences having a number of nucleotidealterations when compared to the starting nucleotide sequences. Geneshuffling (also known as DNA shuffling) involves one or more cycles ofrandom fragmentation and reassembly of the nucleotide sequences,followed by screening to select nucleotide sequences encodingpolypeptides with desired properties. In order for homology-basednucleic acid shuffling to take place, the relevant parts of thenucleotide sequences are preferably at least 50% identical, such as atleast 60% identical, more preferably at least 70% identical, such as atleast 80% identical. The recombination can be performed in vitro or invivo.

Examples of suitable in vitro gene shuffling methods are disclosed byStemmer et al. (1994), Proc. Natl. Acad. Sci. USA; vol. 91, pp.10747-10751; Stemmer (1994), Nature, vol. 370, pp. 389-391; Smith(1994), Nature vol. 370, pp. 324-325; Zhao et al., Nat. Biotechnol.1998, March; 16(3): 258-61; Zhao H. and Arhold, F B, Nucleic AcidsResearch, 1997, Vol. 25. No. 6 pp. 1307-1308; Shao et al., Nucleic AcidsResearch 1998, Jan. 15; 26(2): pp. 681-83; and WO 95/17413.

An example of a suitable in vivo shuffling method is disclosed in WO97/07205. Other techniques for mutagenesis of nucleic acid sequences byin vitro or in vivo recombination are disclosed e.g., in WO 97/20078 andU.S. Pat. No. 5,837,458. Examples of specific shuffling techniquesinclude “family shuffling,” “synthetic shuffling” and “in silicoshuffling”.

Family shuffling involves subjecting a family of homologous genes fromdifferent species to one or more cycles of shuffling and subsequentscreening or selection. Family shuffling techniques are disclosed e.g.,by Crameri et al. (1998), Nature, vol. 391, pp. 288-291; Christians etal. (1999), Nature Biotechnology, vol. 17, pp. 259-264; Chang et al.(1999), Nature Biotechnology, vol. 17, pp. 793-797; and Ness et al.(1999), Nature Biotechnology, vol. 17, 893-896.

Synthetic shuffling involves providing libraries of overlappingsynthetic oligonucleotides based e.g., on a sequence alignment ofhomologous genes of interest. The synthetically generatedoligonucleotides are recombined, and the resulting recombinant nucleicacid sequences are screened and if desired used for further shufflingcycles. Synthetic shuffling techniques are disclosed in WO 00/42561.

In silico shuffling refers to a DNA shuffling procedure, which isperformed or modelled using a computer system, thereby partly orentirely avoiding the need for physically manipulating nucleic acids.Techniques for in silico shuffling are disclosed in WO 00/42560.

Once assembled (by synthesis, site-directed mutagenesis or anothermethod), the nucleotide sequence encoding the polypeptide is insertedinto a recombinant vector and operably linked to control sequencesnecessary for expression of the FVII in the desired transformed hostcell.

It should of course be understood that not all vectors and expressioncontrol sequences function equally well to express the nucleotidesequence encoding a polypeptide described herein. Neither will all hostsfunction equally well with the same expression system. However, one ofskill in the art can make a selection among these vectors, expressioncontrol sequences and hosts without undue experimentation. For example,in selecting a vector, the host must be considered because the vectormust replicate in it or be able to integrate into the chromosome. Thevector's copy number, the ability to control that copy number, and theexpression of any other proteins encoded by the vector, such asantibiotic markers, should also be considered. In selecting anexpression control sequence, a variety of factors should also beconsidered. These include, for example, the relative strength of thesequence, its controllability, and its compatibility with the nucleotidesequence encoding the polypeptide, particularly as regards potentialsecondary structures. Hosts should be selected by consideration of theircompatibility with the chosen vector, the toxicity of the product codedfor by the nucleotide sequence, their secretion characteristics, theirability to fold the polypeptide correctly, their fermentation or culturerequirements, and the ease of purification of the products coded for bythe nucleotide sequence.

The recombinant vector can be an autonomously replicating vector, i.e.,a vector, which exists as an extrachromosomal entity, the replication ofwhich is independent of chromosomal replication, e.g., a plasmid.Alternatively, the vector is one which, when introduced into a hostcell, is integrated into the host cell genome and replicated togetherwith the chromosome(s) into which it has been integrated.

The vector is preferably an expression vector, in which the nucleotidesequence encoding the polypeptide of the invention is operably linked toadditional segments required for transcription of the nucleotidesequence. The vector is typically derived from plasmid or viral DNA. Anumber of suitable expression vectors for expression in the host cellsmentioned herein are commercially available or described in theliterature. Useful expression vectors for eukaryotic hosts, include, forexample, vectors comprising expression control sequences from SV40,bovine papilloma virus, adenovirus and cytomegalovirus. Specific vectorsare, e.g., pCDNA3.1(+)\Hyg (Invitrogen, Carlsbad, Calif., USA) andpCI-neo (Stratagene, La Jola, Calif., USA). Useful expression vectorsfor yeast cells include the 2μ plasmid and derivatives thereof, the POT1vector (U.S. Pat. No. 4,931,373), the pJSO37 vector described in Okkels,Ann. New York Acad. Sci. 782, 202-207, 1996, and pPICZ A, B or C(Invitrogen). Useful vectors for insect cells include pVL941, pBG311(Cate et al., “Isolation of the Bovine and Human Genes for MullerianInhibiting Substance And Expression of the Human Gene In Animal Cells,”Cell, 45, pp. 685-98 (1986), pBluebac 4.5 and pMelbac (both availablefrom Invitrogen). Useful expression vectors for bacterial hosts includeknown bacterial plasmids, such as plasmids from E. coli, includingpBR322, pET3a and pET12a (both from Novagen Inc., Wis., USA), wider hostrange plasmids, such as RP4, phage DNAs, e.g., the numerous derivativesof phage lambda, e.g., NM989, and other DNA phages, such as M13 andfilamentous single stranded DNA phages.

Other vectors for use in this invention include those that allow thenucleotide sequence encoding the polypeptide to be amplified in copynumber. Such amplifiable vectors are well known in the art. Theyinclude, for example, vectors able to be amplified by DHFR amplification(see, e.g., Kaufman, U.S. Pat. No. 4,470,461, Kaufman and Sharp,“Construction of A Modular Dihydrafolate Reductase cDNA Gene: AnalysisOf Signals Utilized For Efficient Expression,” Mol. Cell. Biol., 2, pp.1304-19 (1982)) and glutamine synthetase (“GS”) amplification (see,e.g., U.S. Pat. No. 5,122,464 and EP 338,841).

The recombinant vector can further comprise a DNA sequence enabling thevector to replicate in the host cell in question. An example of such asequence (when the host cell is a mammalian cell) is the SV40 origin ofreplication. When the host cell is a yeast cell, suitable sequencesenabling the vector to replicate are the yeast plasmid 2μ replicationgenes REP 1-3 and origin of replication.

The vector can also comprise a selectable marker, e.g., a gene theproduct of which complements a defect in the host cell, such as the genecoding for dihydrofolate reductase (DHFR) or the Schizosaccharomycespombe TPI gene (described by P. R. Russell, Gene 40, 1985, pp. 125-130),or one which confers resistance to a drug, e.g., ampicillin, kanamycin,tetracyclin, chloramphenicol, neomycin, hygromycin or methotrexate. ForSaccharomyces cerevisiae, selectable markers include ura3 and leu2. Forfilamentous fungi, selectable markers include amdS, pyrg, arcB, niaD andsC.

The term “control sequences” is defined herein to include allcomponents, which are necessary or advantageous for the expression ofthe polypeptide of the invention. Each control sequence can be native orforeign to the nucleic acid sequence encoding the polypeptide. Suchcontrol sequences include, but are not limited to, a leader sequence,polyadenylation sequence, propeptide sequence, promoter, enhancer orupstream activating sequence, signal peptide sequence, and transcriptionterminator. At a minimum, the control sequences include a promoter.

A wide variety of expression control sequences can be used in thepresent invention. Such useful expression control sequences include theexpression control sequences associated with structural genes of theforegoing expression vectors as well as any sequence known to controlthe expression of genes of prokaryotic or eukaryotic cells or theirviruses, and various combinations thereof.

Examples of suitable control sequences for directing transcription inmammalian cells include the early and late promoters of SV40 andadenovirus, e.g., the adenovirus 2 major late promoter, the MT-1(metallothionein gene) promoter, the human cytomegalovirusimmediate-early gene promoter (CMV), the human elongation factor 1α(EF-1α) promoter, the Drosophila minimal heat shock protein 70 promoter,the Rous Sarcoma Virus (RSV) promoter, the human ubiquitin C (UbC)promoter, the human growth hormone terminator, SV40 or adenovirus Elbregion polyadenylation signals and the Kozak consensus sequence (Kozak,M. J Mol Biol 1987 Aug. 20; 196(4):947-50).

In order to improve expression in mammalian cells a synthetic intron canbe inserted in the 5′ untranslated region of the nucleotide sequenceencoding the polypeptide. An example of a synthetic intron is thesynthetic intron from the plasmid pCI-Neo (available from PromegaCorporation, Wis., USA).

Examples of suitable control sequences for directing transcription ininsect cells include the polyhedrin promoter, the P10 promoter, theAutographa californica polyhedrosis virus basic protein promoter, thebaculovirus immediate early gene 1 promoter and the baculovirus 39Kdelayed-early gene promoter, and the SV40 polyadenylation sequence.Examples of suitable control sequences for use in yeast host cellsinclude the promoters of the yeast α-mating system, the yeast triosephosphate isomerase (TPI) promoter, promoters from yeast glycolyticgenes or alcohol dehydrogenase genes, the ADH2-4c promoter, and theinducible GAL promoter. Examples of suitable control sequences for usein filamentous fungal host cells include the ADH3 promoter andterminator, a promoter derived from the genes encoding Aspergillusoryzae TAKA amylase triose phosphate isomerase or alkaline protease, anA. niger α-amylase, A. niger or A. nidulans glucoamylase, A. nidulansacetamidase, Rhizomucor miehei aspartic proteinase or lipase, the TPI1terminator and the ADH3 terminator. Examples of suitable controlsequences for use in bacterial host cells include promoters of the lacsystem, the trp system, the TAC or TRC system, and the major promoterregions of phage lambda.

The presence or absence of a signal peptide will, e.g., depend on theexpression host cell used for the production of the polypeptide to beexpressed (whether it is an intracellular or extracellular polypeptide)and whether it is desirable to obtain secretion. For use in filamentousfungi, the signal peptide can conveniently be derived from a geneencoding an Aspergillus sp. amylase or glucoamylase, a gene encoding aRhizomucor miehei lipase or protease or a Humicola lanuginosa lipase.The signal peptide is preferably derived from a gene encoding A. oryzaeTAKA amylase, A. niger neutral α-amylase, A. niger acid-stable amylase,or A. niger glucoamylase. For use in insect cells, the signal peptidecan conveniently be derived from an insect gene (cf. WO 90/05783), suchas the Lepidopteran manduca sexta adipokinetic hormone precursor, (cf.U.S. Pat. No. 5,023,328), the honeybee meiittin (Invitrogen),ecdysteroid UDPglucosyltransferase (egt) (Murphy et al., ProteinExpression and Purification 4, 349-357 (1993) or human pancreatic lipase(hpl) (Methods in Enzymology 284, pp. 262-272, 1997). A preferred signalpeptide for use in mammalian cells is that of hFVII or the murine Igkappa light chain signal peptide (Coloma, M (1992) J. Imm. Methods152:89-104). For use in yeast cells suitable signal peptides have beenfound to be the α-factor signal peptide from S. cereviciae (cf. U.S.Pat. No. 4,870,008), a modified carboxypeptidase signal peptide (cf. L.A. Valls et al., Cell 48, 1987, pp. 887-897), the yeast BAR1 signalpeptide (cf. WO 87/02670), the yeast aspartic protease 3 (YAP3) signalpeptide (cf. M. Egel-Mitani et al., Yeast 6, 1990, pp. 127-137), and thesynthetic leader sequence TA57 (WO98/32867). For use in E. coli cells asuitable signal peptide have been found to be the signal peptide ompA(EP581821).

The nucleotide sequence of the invention encoding a FVII polypeptide,whether prepared by site-directed mutagenesis, synthesis, PCR or othermethods, can optionally include a nucleotide sequence that encode asignal peptide. The signal peptide is present when the polypeptide is tobe secreted from the cells in which it is expressed. Such signalpeptide, if present, should be one recognized by the cell chosen forexpression of the polypeptide. The signal peptide can be homologous(e.g., be that normally associated with hFVII) or heterologous (i.e.,originating from another source than hFVII) to the polypeptide or can behomologous or heterologous to the host cell, i.e., be a signal peptidenormally expressed from the host cell or one which is not normallyexpressed from the host cell. Accordingly, the signal peptide can beprokaryotic, e.g., derived from a bacterium such as E. coli, oreukaryotic, e.g., derived from a mammalian, or insect or yeast cell.

Any suitable host can be used to produce the polypeptide or polypeptidepart of the conjugate of the invention, including bacteria, fungi(including yeasts), plant, insect, mammal, or other appropriate animalcells or cell lines, as well as transgenic animals or plants. Examplesof bacterial host cells include grampositive bacteria such as strains ofBacillus, e.g., B. brevis or B. subtilis, Pseudomonas or Streptomyces,or gramnegative bacteria, such as strains of E. coli. The introductionof a vector into a bacterial host cell can, for instance, be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, MolecularGeneral Genetics 168: 111-115), using competent cells (see, e.g.; Youngand Spizizin, 1961, Journal of Bacteriology 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, Journal of Molecular Biology 56: 209-221),electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6:742-751), or conjugation (see, e.g., Koehler and Thorne, 1987, Journalof Bacteriology 169: 5771-5278). Examples of suitable filamentous fungalhost cells include strains of Aspergillus, e.g., A. oryzae, A. niger, orA. nidulans, Fusarium or Trichoderma. Fungal cells can be transformed bya process involving protoplast formation, transformation of theprotoplasts, and regeneration of the cell wall in a manner known per se.Suitable procedures for transformation of Aspergillus host cells aredescribed in EP 238 023 and U.S. Pat. No. 5,679,543. Suitable methodsfor transforming Fusarium species are described by Malardier et al.,1989, Gene 78: 147-156 and WO 96/00787. Examples of suitable yeast hostcells include strains of Saccharomyces, e.g., S. cerevisiae,Schizosaccharomyces, Klyveromyces, Pichia, such as P. pastoris or P.methanolica, Hansenula, such as H. Polymorpha or Yarrowia. Yeast can betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, Journal ofBacteriology 153: 163; Hinnen et al., 1978, Proceedings of the NationalAcademy of Sciences USA 75: 1920: and as disclosed by ClontechLaboratories, Inc, Palo Alto, Calif., USA (in the product protocol forthe Yeastmaker™ Yeast Transformation System Kit). Examples of suitableinsect host cells include a Lepidoptora cell line, such as Spodopterafrugiperda (Sf9 or Sf21) or Trichoplusioa ni cells (High Five) (U.S.Pat. No. 5,077,214). Transformation of insect cells and production ofheterologous polypeptides therein can be performed as described byInvitrogen. Examples of suitable mammalian host cells include Chinesehamster ovary (CHO) cell lines, (e.g., CHO-K1; ATCC CCL-61), GreenMonkey cell lines (COS) (e.g., COS 1 (ATCC CRL-1650), COS 7 (ATCCCRL-1651)); mouse cells (e.g., NS/O), Baby Hamster Kidney (BHK) celllines (e.g., ATCC CRL-1632 or ATCC CCL-10), and human cells (e.g., HEK293 (ATCC CRL-1573)), as well as plant cells in tissue culture.Additional suitable cell lines are known in the art and available frompublic depositories such as the American Type Culture Collection,Rockville, Md. Also, the mammalian cell, such as a CHO cell, can bemodified to express sialyltransferase, e.g., 1,6-sialyltransferase,e.g., as described in U.S. Pat. No. 5,047,335, in order to provideimproved glycosylation of the FVII or FVIIa polypeptide.

In order to increase secretion it can be of particular interest toproduce the polypeptide of the invention together with an endoprotease,in particular a PACE (Paired basic amino acid converting enzyme) (e.g.,as described in U.S. Pat. No. 5,986,079), such as a Kex2 endoprotease(e.g., as described in WO 00/28065).

Methods for introducing exogeneous DNA into mammalian host cells includecalcium phosphate-mediated transfection, electroporation, DEAE-dextranmediated transfection, liposome-mediated transfection, viral vectors andthe transfection method described by Life Technologies Ltd, Paisley, UKusing Lipofectamin 2000. These methods are well known in the art ande.g., described by Ausbel et al. (eds.), 1996, Current Protocols inMolecular Biology, John Wiley & Sons, New York, USA. The cultivation ofmammalian cells are conducted according to established methods, e.g., asdisclosed in (Animal Cell Biotechnology, Methods and Protocols, Editedby Nigel Jenkins, 1999, Human Press Inc, Totowa, N.J., USA and HarrisonM A and Rae I F, General Techniques of Cell Culture, CambridgeUniversity Press 1997).

In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods known in the art. For example, the cell can becultivated by shake flask cultivation, small-scale or large-scalefermentation (including continuous, batch, fed-batch, or solid statefermentations) in laboratory or industrial fermenters performed in asuitable medium and under conditions allowing the polypeptide to beexpressed and/or isolated. The cultivation takes place in a suitablenutrient medium comprising carbon and nitrogen sources and inorganicsalts, using procedures known in the art. Suitable media are availablefrom commercial suppliers or can be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The resulting polypeptide can be recovered by methods known in the art.For example, the polypeptide can be recovered from the nutrient mediumby conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray drying, evaporation, orprecipitation.

The polypeptides can be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Jansonand Lars Ryden, editors, VCH Publishers, New York, 1989).

Single chain FVII can be purified and activated to two-chain FVIIa by anumber of methods as described in the literature (Broze and Majerus,1980, J. Biol. Chem. 255:1242-47 and Hedner and Kisiel, 1983, J. Clin.Invest. 71:1836-41). Another method whereby single chain FVII can bepurified is by incorporation of Zn ions during purification as describedin U.S. Pat. No. 5,700,914.

In a preferred embodiment, the polypeptide is purified as a single chainFVII, which further is PEGylated. The PEGylated FVII single chainpolypeptide is activated by either use of an immobilized enzyme (e.g.,factors IIa, IXa, Xa and XIIa) or by autoactivation using a positivelycharged ion exchange matrix or the like.

It is advantageous to first purify FVII in its single chain form, thenPEGylate (if desired) and last activate by one of the methods describedabove or by autoactivation as described by Pedersen et al, 1989,Biochemistry 28: 9331-36. The advantage of carrying out PEGylationbefore activation is that PEGylation of the new aminoterminal formed bycleavage of R152-I153 is avoided. PEGylation of this new amino terminalwould render the molecule inactive since the formation of a hydrogenbond between D242 and the amino terminal of I153 is necessary foractivity.

Pharmaceutical Composition of the Invention and its Use

In a further aspect, the present invention relates to a composition, inparticular to a pharmaceutical composition, comprising a polypeptide orconjugate (including an inactive conjugate as described further above)of the invention and a pharmaceutically acceptable carrier or excipient.

The conjugate, the polypeptide or the pharmaceutical compositionaccording to the invention can be used as a medicament.

Preferably, the polypeptide or the (active) conjugate can be used forthe manufacture of a medicament for the treatment or prophylaxis of aFVIIa/TF-related disease or disorder in a mammal. For example, thepolypeptide or the (active) conjugate can be used for the manufacture ofa medicament for the treatment or prophylaxis of diseases whereinincreased clot formation is desirable, such as treatment of patientshaving diseases resulting in inadequate blood coagulation in response todamage to blood vessels. In particular, the polypeptide or the (active)conjugate can be used for the manufacture of a medicament for thetreatment of hemophiliacs, hemophiliacs with inhibitors to FVIII andFIX, patients with thrombocytopenia, patients with thrombocytopathies,such as Glanzmann's thrombastenia platelet release defects and storagepool defects, patients with von Willebrand's disease, patients withliver diseases, or otherwise healthy people with severe bleedingproblems, e.g., due to trauma or major surgery, who have developedinhibitors to FVIIa, bleeding disorders such as hemophiliacs and othertypically associated with severe tissue damages.

Analogously, the inactive conjugate of the invention can be used for themanufacture of a medicament for the treatment or prophylaxis of aFVIIa/TF-related disease or disorder in a mammal. For example, theinactive conjugate of the invention can be used for the manufacture of amedicament for the treatment or prophylaxis of diseases where decreasedclot formation is desirable, such as prophylaxis or treatment ofpatients being in hypercoagulable states, such as patients with sepsis,deep-vein thrombosis, patients in risk of myocardial infections orthrombotic stroke, pulmonary embolism, patients with acute coronarysyndromes (myocardial infarction and unstable angina pectoris), patientsundergoing coronary cardiac, prevention of cardiac events and restonosisfor patients receiving angioplasty, patients with peripheral vasculardiseases. The inactive conjugate of the invention can also be used forthe manufacture of a medicament for the treatment of respiratorydiseases, tumor growth and metastasis.

In another aspect, the polypeptide, the (active) conjugate or thepharmaceutical composition comprising the (active) conjugate of theinvention can be used in a method for treating a mammal having aFVIIa/TF-related disease or disorder (such as one or more of thediseases or disorders mentioned above), comprising administering to amammal in need thereof an effective amount of such a polypeptide,conjugate or composition.

Analogously, the inactive conjugate or the pharmaceutical compositioncomprising the inactive conjugate of the invention can be used in amethod for treating a mammal having a FVIIa/TF-related disease ordisorder (such as one or more of the diseases or disorders mentionedabove), comprising administering to a mammal in need thereof aneffective amount of such an inactivated conjugate or composition.

The polypeptides or conjugates of the invention is administered topatients in a therapeutically effective dose, normally one approximatelyparalleling that employed in therapy with rFVII such as NovoSeven®, orat higher dosage. By “therapeutically effective dose” herein is meant adose that is sufficient to produce the desired effects in relation tothe condition for which it is administered. The exact dose will dependon the circumstances, and will be ascertainable by one skilled in theart using known techniques. Normally, the dose should be capable ofpreventing or lessening the severity or spread of the condition orindication being treated. It will be apparent to those of skill in theart that an effective amount of a polypeptide, conjugate or compositionof the invention depends, inter alia, upon the disease, the dose, theadministration schedule, whether the polypeptide or conjugate orcomposition is administered alone or in conjunction with othertherapeutic agents, the plasma half-life of the compositions, and thegeneral health of the patient. Preferably, the polypeptide, conjugate,or composition of the invention is administered in an effective dose, inparticular a dose which is sufficient to normalize the coagulationdisorder.

The polypeptide or conjugate of the invention is preferably administeredin a composition including a pharmaceutically acceptable carrier orexcipient. “Pharmaceutically acceptable” means a carrier or excipientthat does not cause any untoward effects in patients to whom it isadministered. Such pharmaceutically acceptable carriers and excipientsare well known in the art (see, for example, Remington's PharmaceuticalSciences, 18th edition, A. R. Gennaro, Ed., Mack Publishing Company[1990]; Pharmaceutical Formulation Development of Peptides and Proteins,S. Frokjaer and L. Hovgaard, Eds., Taylor & Francis [2000]; and Handbookof Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., PharmaceuticalPress [2000]).

The polypeptide or conjugate of the invention can be formulated intopharmaceutical compositions by well-known methods. Suitable formulationsare described by Remington's Pharmaceutical Sciences by E. W. Martin(Mark Publ. Co., 16th Ed., 1980).

The polypeptide or conjugate of the invention can be used “as is” and/orin a salt form thereof. Suitable salts include, but are not limited to,salts with alkali metals or alkaline earth metals, such as sodium,potassium, calcium and magnesium, as well as e.g., zinc salts. Thesesalts or complexes can be present as a crystalline and/or amorphousstructure.

The pharmaceutical composition of the invention can be administeredalone or in conjunction with other therapeutic agents. These agents canbe incorporated as part of the same pharmaceutical composition or can beadministered separately from the polypeptide or conjugate of theinvention, either concurrently or in accordance with another treatmentschedule. In addition, the polypeptide, conjugate or pharmaceuticalcomposition of the invention can be used as an adjuvant to othertherapies.

A “patient” for the purposes of the present invention includes bothhumans and other mammals. Thus the methods are applicable to both humantherapy and veterinary applications.

The pharmaceutical composition of the polypeptide or conjugate of theinvention can be formulated in a variety of forms, e.g., as a liquid,gel, lyophilized, or as a compressed solid. The preferred form willdepend upon the particular indication being treated and will be apparentto one skilled in the art.

In particular, the pharmaceutical composition of the polypeptide orconjugate of the invention can be formulated in lyophilised or stablesoluble form. The polypeptide or the conjugate can be lyophilised by avariety of procedures known in the art. A polypeptide or the conjugatecan be a stable soluble form by the removal or shielding of proteolyticdegradation sites. The advantage of obtaining a stable solublepreparation lies in easier handling for the patient and, in the case ofemergencies, quicker action, which potentially can become life saving.The preferred form will depend upon the particular indication beingtreated and will be apparent to one of skill in the art.

The administration of the formulations of the present invention can beperformed in a variety of ways, including, but not limited to, orally,subcutaneously, intravenously, intracerebrally, intranasally,transdermally, intraperitoneally, intramuscularly, intrapulmonary,vaginally, rectally, intraocularly, or in any other acceptable manner.The formulations can be administered continuously by infusion, althoughbolus injection is acceptable, using techniques well known in the art,such as pumps or implantation. In some instances the formulations can bedirectly applied as a solution or spray.

Parenterals

A preferred example of a pharmaceutical composition is a solutiondesigned for parenteral administration. Although in many casespharmaceutical solution formulations are provided in liquid form,appropriate for immediate use, such parenteral formulations can also beprovided in frozen or in lyophilized form. In the former case, thecomposition must be thawed prior to use. The latter form is often usedto enhance the stability of the active compound contained in thecomposition under a wider variety of storage conditions, as it isrecognized by those skilled in the art that lyophilized preparations aregenerally more stable than their liquid counterparts. Such lyophilizedpreparations are reconstituted prior to use by the addition of one ormore suitable pharmaceutically acceptable diluents such as sterile waterfor injection or sterile physiological saline solution.

In case of parenterals, they are prepared for storage as lyophilizedformulations or aqueous solutions by mixing, as appropriate, thepolypeptide having the desired degree of purity with one or morepharmaceutically acceptable carriers, excipients or stabilizerstypically employed in the art (all of which are termed “excipients”),for example buffering agents, stabilizing agents, preservatives,isotonifiers, non-ionic surfactants or detergents, antioxidants and/orother miscellaneous additives.

Buffering agents help to maintain the pH in the range which approximatesphysiological conditions. They are typically present at a concentrationranging from about 2 mM to about 50 mM Suitable buffering agents for usewith the present invention include both organic and inorganic acids andsalts thereof such as citrate buffers (e.g., monosodium citrate-disodiumcitrate mixture, citric acid-trisodium citrate mixture, citricacid-monosodium citrate mixture, etc.), succinate buffers (e.g.,succinic acid-monosodium succinate mixture, succinic acid-sodiumhydroxide mixture, succinic acid-disodium succinate mixture, etc.),tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaricacid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture,etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture,fumaric acid-disodium fumarate mixture, monosodium fumarate-disodiumfumarate mixture, etc.), gluconate buffers (e.g., gluconic acid-sodiumglyconate mixture, gluconic acid-sodium hydroxide mixture, gluconicacid-potassium glyuconate mixture, etc.), oxalate buffer (e.g., oxalicacid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture,oxalic acid-potassium oxalate mixture, etc.), lactate buffers (e.g.,lactic acid-sodium lactate mixture, lactic acid-sodium hydroxidemixture, lactic acid-potassium lactate mixture, etc.) and acetatebuffers (e.g., acetic acid-sodium acetate mixture, acetic acid-sodiumhydroxide mixture, etc.). Additional possibilities are phosphatebuffers, histidine buffers and trimethylamine salts such as Tris.

Stabilizers refer to a broad category of excipients which can range infunction from a bulking agent to an additive which solubilizes thetherapeutic agent or helps to prevent denaturation or adherence to thecontainer wall. Typical stabilizers can be polyhydric sugar alcohols(enumerated above); amino acids such as arginine, lysine, glycine,glutamine, asparagine, histidine, alanine, omithine, L-leucine,2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugaralcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol,xylitol, ribitol, myoinisitol, galactitol, glycerol and the like,including cyclitols such as inositol; polyethylene glycol; amino acidpolymers; sulfur-containing reducing agents, such as urea, glutathione,thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglyceroland sodium thiosulfate; low molecular weight polypeptides (i.e., <10residues); proteins such as human serum albumin, bovine serum albumin,gelatin or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructoseand glucose; disaccharides such as lactose, maltose and sucrose;trisaccharides such as raffinose, and polysaccharides such as dextran.Stabilizers are typically present in the range of from 0.1 to 10,000parts by weight based on the active protein weight.

Preservatives are added to retard microbial growth, and are typicallyadded in amounts of about 0.2%-1% (w/v). Suitable preservatives for usewith the present invention include phenol, benzyl alcohol, meta-cresol,methyl paraben, propyl paraben, octadecyldimethylbenzyl ammoniumchloride, benzalkonium halides (e.g., benzalkonium chloride, bromide oriodide), hexamethonium chloride, alkyl parabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol and 3-pentanol.

Isotonicifiers are added to ensure isotonicity of liquid compositionsand include polyhydric sugar alcohols, preferably trihydric or highersugar alcohols, such as glycerin, erythritol, arabitol, xylitol,sorbitol and mannitol. Polyhydric alcohols can be present in an amountbetween 0.1% and 25% by weight, typically 1% to 5%, taking into accountthe relative amounts of the other ingredients.

Non-ionic surfactants or detergents (also known as “wetting agents”) canbe present to help solubilizing the therapeutic agent as well as toprotect the therapeutic polypeptide against agitation-inducedaggregation, which also permits the formulation to be exposed to shearsurface stress without causing denaturation of the polypeptide. Suitablenon-ionic surfactants include polysorbates (20, 80, etc.), polyoxamers(184, 188 etc.), Pluronic® polyols, polyoxyethylene sorbitan monoethers(Tween®-20, Tween®)-80, etc.).

Additional miscellaneous excipients include bulking agents or fillers(e.g., starch), chelating agents (e.g., EDTA), antioxidants (e.g.,ascorbic acid, methionine, vitamin E) and cosolvents.

The active ingredient can also be entrapped in microcapsules prepared,for example, by coascervation techniques or by interfacialpolymerization, for example hydroxymethylcellulose, gelatin orpoly-(methylmethacylate) microcapsules, in colloidal drug deliverysystems (for example liposomes, albumin microspheres, microemulsions,nano-particles and nanocapsules) or in macroemulsions. Such techniquesare disclosed in Remington's Pharmaceutical Sciences, supra.

Parenteral formulations to be used for in vivo administration must besterile. This is readily accomplished, for example, by filtrationthrough sterile filtration membranes.

Sustained Release Preparations

Examples of sustained-release preparations include semi-permeablematrices of solid hydrophobic polymers containing the polypeptide orconjugate, the matrices having a suitable form such as a film ormicrocapsules. Examples of sustained-release matrices includepolyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate) orpoly(vinylalcohol)), polylactides, copolymers of L-glutamic acid andethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the ProLease® technology orLupron Depot® (injectable microspheres composed of lactic acid-glycolicacid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyricacid. While polymers such as ethylene-vinyl acetate and lacticacid-glycolic acid enable release of molecules for long periods such asup to or over 100 days, certain hydrogels release proteins for shortertime periods. When encapsulated polypeptides remain in the body for along time, they can denature or aggregate as a result of exposure tomoisture at 37° C., resulting in a loss of biological activity andpossible changes in immunogenicity. Rational strategies can be devisedfor stabilization depending on the mechanism involved. For example, ifthe aggregation mechanism is discovered to be intermolecular S—S bondformation through thio-disulfide interchange, stabilization can beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

The invention is further described in the following non-limitingexamples.

MATERIALS AND METHODS

Methods Used to Determine the Amino Acids to be Modified

Accessible Surface Area (ASA)

The computer program Access (B. Lee and F. M. Richards, J. Mol. Biol.55: 379-400 (1971)) version 2 (© 1983 Yale University) are used tocompute the accessible surface area (ASA) of the individual atoms in thestructure. This method typically uses a probe-size of 1.4 Å and definesthe Accessible Surface Area (ASA) as the area formed by the center ofthe probe. Prior to this calculation all water molecules and allhydrogen atoms should be removed from the coordinate set, as shouldother atoms not directly related to the protein.

Fractional ASA of Side Chain

The fractional ASA of the side chain atoms is computed by division ofthe sum of the ASA of the atoms in the side chain with a valuerepresenting the ASA of the side chain atoms of that residue type in anextended Ala-x-Ala tripeptide (See Hubbard, Campbell & Thornton (1991)J. Mol. Biol. 220, 507-530). For this example the CA atom is regarded asa part of the side chain of Glycine residues but not for the remainingresidues. The following table is used as standard 100% ASA for the sidechain:

Ala  69.23 Å² Arg 200.35 Å² Asn 106.25 Å² Asp 102.06 Å² Cys  96.69 Å²Gln 140.58 Å² Glu 134.61 Å² Gly  32.28 Å² His 147.00 Å² Ile 137.91 Å²Leu 140.76 Å² Lys 162.50 Å² Met 156.08 Å² Phe 163.90 Å² Pro 119.65 Å²Ser  78.16 Å² Thr 101.67 Å² Trp 210.89 Å² Tyr 176.61 Å² Val 114.14 Å²

Residues not detected in the structure are defined as having 100%exposure as they are thought to reside in flexible regions. Thegamma-carboxy glutamic acids at positions 6, 7, 14, 16, 19, 20, 25, 26,29 and 35 are all defined as being 100% exposed.

Determining Distances Between Atoms

The distance between atoms is most easily determined using moleculargraphics software e.g., InsightII® v. 98.0, MSI INC.

Catalytic Site Region

The catalytic site region is defined as any residues having at least oneatom within 10 Å of any atom in the catalytic triad (residues H193,D242, S344).

Determination of Tissue Factor Binding Site

The receptor-binding site is defined as comprising of all residueshaving their accessible surface area changed upon receptor binding. Thisis determined by at least two ASA calculations; one on the isolatedligand(s) in the ligand(s)/receptor(s) complex and one on the completeligand(s)/receptor(s) complex.

Methods for Testing FVII and FVIIa Properties

Measurement of Functional In Vivo Half-Life

Measurement of in vivo biological half-life can be carried out in anumber of ways as described in the literature. An example of an assayfor the measurement of in vivo half-life of rFVIIa or variants thereofis described in FDA reference number 96-0597. Briefly, FVII clottingactivities are measured in plasma drawn prior to and during a 24-hourperiod after administration of the conjugate, polypeptide orcomposition. The median apparent volume of distribution at steady stateis measured and the median clearance determined.

Measurement of Reduced Sensitivity to Proteolytic Degradation

A composition containing the conjugate (100-750 μg/ml, preferably 600μg/ml), 1.5 mg Ca²⁺/ml (as calcium chloride), mannitol (30 mg/ml),polysorbate 80 (0.1 mg/ml), sodium chloride (3 mg/ml) and glycyl-glycinebuffer (1.3 mg/ml, pH 5.5) is prepared.

A similar composition containing wild-type rFVIIa is prepared.

The initial clotting activity or, alternatively, the initial amidolyticactivity is then determined as described in the section entitled “Methodfor measuring the clotting activity” or as described in the sectionentitled “Method of measuring low levels of catalytic activity” or“Method of measuring the catalytic activity.”

The compositions are then incubated at 37° C. until the compositioncontaining the wild-type rFVII has lost at least 25%, preferably atleast 50%, of its initial clotting or amidolytic acitivity.

The clotting or amidolytic activity of the composition containing theconjugate of the invention is then measured.

The reduced sensitivity to proteolytic degradation of the conjugate ofthe invention as compared to wild-type rFVIIa is then expressed inpercentage.

Alternative Methods for Measuring Reduced Sensitivity to ProteolyticDegradation

Proteolytic degradation can be measured using the assay described inU.S. Pat. No. 5,580,560, Example 5, where proteolysis isautoproteolysis.

Furthermore, reduced proteolysis can be tested in an in vivo model usingradiolabelled samples and comparing proteolysis of wild type andconjugates by withdrawing blood samples and subjecting these to SDS-PAGEand autoradiography.

Irrespectively of the assay used for determining proteolyticdegradation, “reduced proteolytic degradation” is intended to mean ameasurable reduction in cleavage compared to that obtained bynon-conjugated wild type FVIIa as measured by gel scanning of Coomassiestained SDS-PAGE gels, HPLC or as measured by conserved catalyticactivity in comparison to wild type using the chromogenic assaydescribed below.

Determination of the Molecular Weight of rFVII and Conjugates Thereof

The molecular weight of conjugated or unconjugated rFVII or conjugatesthereof is determined by either SDS-PAGE, gel filtration, Western Blots,matrix assisted laser desorption mass spectrometry or equilibriumcentrifugation, e.g., SDS-PAGE according to Laemmli, U.K., Nature Vol227 (1970), pp. 680-85.

Method of Measuring Low Levels of Catalytic Activity

The amidolytic activity in dilute samples of FVII/FVIIa and fermentationliquid/conditioned medium can be determined using COASET® FVII(Chromogenix, Art. No 82 19 00). The amidolytic activity is determinedin accordance with the manufacturer's instructions. Briefly, FX ispresent in surplus and converted to FXa by FVIIa at 37° C. The generatedFXa then hydrolyses the chromogenic substrate S2765(N-α-Cbo-D-Arg-Gly-Arg-pNA) resulting in liberation of the chromophoricmolecule, para-nitro-anillin (pNA) absorbing light at wavelength 405 nm.The reaction is stopped by addition of acetic acid. The amount ofFVII/FVIIa in the sample is determined by comparison to a standard curveprepared from FVIIa (ranging from 125 pg/ml to 1 ng/ml in assay buffer).

Method of Measuring the Catalytic Activity

The ability of the conjugates to cleave small peptide substrates can bemeasured using the chromogenic substrate S-2288(D-Ile-Pro-Arg-p-nitroanilide). Recombinant FVIIa is diluted in 0.1 MTris, 0.1 M NaCl, 5 mM CaCl₂, pH 8.3 containing 0.1% BSA. The reactionis started by addition of the S-2288 substrate to 1 mM and theabsorption at 405 nm is measured after incubation for 30 min. at 37° C.

Method of Measuring the Clotting Activity

FVIIa activity is measured using a standard one-stage clotting assayessentially as described in WO92/15686. Briefly, the sample to be testedis diluted in 50 mM Tris (pH 7.5), 0.1% BSA and 100 μl is incubated with100 μl of FVII deficient plasma and 200 μl of thromboplastin Ccontaining 10 mM Ca⁺⁺. Clotting times are measured and compared to astandard curve using a pool of citrated normal human plasma in serialdilution.

Method of Measuring the Anticoagulant Activity

The anticoagulant activity of an inactive FVII or FVIIa conjugate can bemeasured using the one-stage clotting assay described above (Method ofmeasuring the clotting activity) where the inactive conjugate competeswith wild-type FVII for a limited amount of relipidated tissue factor.The assay is performed essentially as described in WO 92/15686, exampleIII, which is hereby incorporated as reference. The ability of theinactive conjugate to prolong the clotting time of wild-type FVII isrecorded and taken as a measure of anticoagulant activity.

EXAMPLES Example 1

The X-ray structure of hFVIIa in complex with soluble tissue factor byBanner et. al., J Mol Biol, 1996; 285:2089 is used for this example. Itis noted that the numbering of residues in the reference does not followthe sequence. Here we have used the sequential numbering according toSEQ ID NO:1. The gamma-carboxy glutamic acids at positions 6, 7, 14, 16,19, 20, 25, 26, 29 and 35 are all here named GLU (three letterabbreviation) or E (one letter abbreviation). Residues 143-152 are notpresent in the structure.

Surface Exposure

Performing fractional ASA calculations on FVII fragments alone combinedwith the definition of accessibilities of non standard and/or missingresidues described in the methods resulted in the following residueshaving more than 25% of their side chain exposed to the surface: A1, N2,A3, F4, L5, E6, E7, L8, R9, P10, S12, L13, E14, E16, K18, E19, E20, Q21,S23, F24, E25, E26, R28, E29, F31, K32, D33, A34, E35, R36, K38, L39,W41, I42, S43, S45, G47, D48, Q49, A51, S52, S53, Q56, G58, S60, K62,D63, Q64, L65, Q66, S67, I69, F71, L73, P74, A75, E77, G78, R79, E82,T83, H84, K85, D86, D87, Q88, L89, I90, V92, N93, E94, G97, E99, S103,D104, H105, T106, G107, T108, K109, S111, R113, E116, G117, S119, L120,L121, A122, D123, G124, V125, S126, T128, P129, T130, V131, E132, I140,L141, E142, K143, R144, N145, A146, S147, K148, P149, Q150, G151, R152,G155, K157, V158, P160, K161, E163, L171, N173, G174, A175, N184, T185,I186, H193, K197, K199, N200, R202, N203, I205, S214, E215, H216, D217,G218, D219, S222, R224, S232, T233, V235, P236, G237, T238, T239, N240,H249, Q250, P251, V253, T255, D256, E265, R266, T267, E270, R271 , F275,V276, R277, F278, L280, L287, L288, D289, R290, G291, A292, T293, L295,E296, N301, M306, T307, Q308, D309, L311, Q312, Q313, R315, K316, V317,G318, D319, S320, P321, N322, T324, E325, Y326, Y332, S333, D334, S336,K337, K341, G342, H351, R353, G354, Q366, G367, T370, V371, G372, R379,E385, Q388, K389, R392, S393, E394, P395, R396, P397, G398, V399, L400,L401, R402, P404 and P406.

The following residues had more than 50% of their side chain exposed tothe surface: A1, A3, F4, L5, E6, E7, L8, R9, P10, E14, E16, K18, E19,E20, Q21, S23, E25, E26, E29, K32, A34, E35, R36, K38, L39, I42, S43,G47, D48, A51, S52, S53, Q56, G58, S60, K62, L65, Q66, S67, I69, F71,L73, P74, A75, E77, G78, R79, E82, H84, K85, D86, D87, Q88, L89, I90,V92, N93, E94, G97, T106, G107, T108, K109, S111, E116, S119, L121,A122, D123, G124, V131, E132, L141, E142, K143, R144, N145, A146, S147,K148, P149, Q150, G151, R152, G155, K157, P160, N173, G174, A175, K197,K199, N200, R202, S214, E215, H216, G218, R224, V235, P236, G237, T238,H249, Q250, V253, D256, T267, F275, R277, F278, L288, D289, R290, G291,A292, T293, L295, N301, M306, Q308, D309, L311, Q312, Q313, R315, K316,G318, D319, N322, E325, D334, K341, G354, G367, V371, E385, K389, R392,E394, R396, P397, G398, R402, P404 and P406.

Tissue Factor Binding Site

Performing ASA calculations the following residues in human FVII changetheir ASA in the complex. These residues were defined as constitutingthe receptor binding site: L13, K18, F31, E35, R36, L39, F40, I42, S43,S60, K62, D63, Q64, L65, I69, C70, F71, C72, L73, P74, F76, E77, G78,R79, E82, K85, Q88, 190, V92, N93, E94, R271, A274, F275, V276, R277,F278, R304, L305, M306, T307, Q308, D309, Q312, Q313, E325 and R379.

Active Site Region

The active site region is defined as any residue having at least oneatom within a distance of 10 Å from any atom in the catalytic triad(residues H193, D242, S344): I153, Q167, V168, L169, L170, L171, Q176,L177, C178, G179, G180, T181, V188, V189, S190, A191, A192, H193, C194,F195, D196, K197, I198, W201, V228, I229, I230, P231, S232, T233, Y234,V235, P236, G237, T238, T239, N240, H241, D242, I243, A244, L245, L246,V281, S282, G283, W284, G285, Q286, T293, T324, E325, Y326, M327, F328,D338, S339, C340, K341, G342, D343, S344, G345, G346, P347, H348, L358,T359, G360, I361, V362, S363, W364, G365, C368, V376, Y377, T378, R379,V380, Q382, Y383, W386, L387, L400, and F405.

The Ridge of the Active Site Binding Cleft

The ridge of the active site binding cleft region was defined by visualinspection of the FVIIa structure 1FAK.pdb as: N173, A175, K199, N200,N203, D289, R290, G291, A292, P321 and T370.

Example 2

Design of an Expression Cassette for Expression of Human BloodCoagulation Factor VII in Mammalian Cells

The DNA sequence shown in SEQ ID NO:2, encompassing the short form ofthe full length cDNA encoding human blood coagulation factor VII withits native short signal peptide (Hagen et al., 1986. PNAS 83:2412), wassynthesized in order to facilitate high expression in mammalian cells.First the ATG start codon context was modified according to the Kozakconsensus sequence (Kozak, M. J Mol Biol 1987 Aug. 20; 196(4):947-50),so that there is a perfect match to the consensus sequence upstream ofthe ATG start codon. Secondly the open reading frame of the native humanblood coagulation factor cDNA was modified by maling a bias in the codonusage towards the codons frequently used in highly expressed humangenes. Further, two translational stop codons was inserted at the end ofthe open reading frame in order to facilitate efficient translationalstop. The fully synthetic and expression optimized human FVII gene wasassembled from 70-mer DNA oligonucleotides and finally amplified usingend primers inserting BamHI and HindIII sites at the 5′ and 3′ endsrespectively using standard PCR techniques, which resulted in thefollowing sequence (SEQ ID NO:4):

ggatcccgccaccatggtcagccaggccctccgcctcctgtgcctgctcctggggctgcagggctgcctggctgccgtcttcgtcacccaggaggaagcccatggcgtcctgcatcgccggcgccgggccaatgcctttctggaagagctccgccctggctccctggaacgcgaatgcaaagaggaacagtgcagctttgaggaagcccgggagattttcaaagacgctgagcggaccaaactgttttggattagctatagcgatggcgatcagtgcgcctccagcccttgccagaacgggggctcctgcaaagaccagctgcagagctatatctgcttctgcctgcctgcctttgaggggcgcaattgcgaaacccataaggatgaccagctgatttgcgtcaacgaaaacgggggctgcgagcagtactgcagcgatcacacgggcacgaagcggagctgccgctgccacgaaggctatagcctcctggctgacggggtgtcctgcacgcccacggtggaatacccttgcgggaagattcccattctagaaaagcggaacgctagcaaaccccagggccggatcgtcggcgggaaggtctgccctaagggggagtgcccctggcaggtcctgctcctggtcaacggggcccagctgtgcggcgggaccctcatcaataccatttgggtcgtgtccgccgctcactgcttcgataagattaagaattggcggaacctcatcgctgtgctcggcgaacacgatctgtccgagcatgacggggacgaacagtcccgccgggtggctcaggtcatcattccctccacctatgtgcctggcacgaccaatcacgatatcgctctgctccgcctccaccagcccgtcgtgctcaccgatcacgtcgtgcctctgtgcctgcctgagcggacctttagcgaacgcacgctggctttcgtccgctttagcctcgtgtccggctggggccagctgctcgaccggggcgctaccgctctcgagctgatggtgctcaacgtcccccggctgatgacccaggactgcctgcagcagtcccgcaaagtgggggactcccccaatatcacggagtatatgttttgcgctggctatagcgatggctccaaggatagctgcaagggggactccggcgggccccatgccacgcactatcgcgggacctggtacctcaccgggatcgtcagctggggccagggctgcgccacggtggggcactttggcgtctacacgcgcgtcagccagtacattgagtggctgcagaagctcatgcggagcgaaccccggcccggggtgctcctgcgggcccctttcccttgata aaagctt

A vector for the cloning of the generated PCR product encompassing theexpression cassette for native human blood coagulation factor VII wasprepared by cloning the intron from pCINeo (Promega). The syntheticintron from pCI-Neo was amplified using standard PCR conditions asdescribed above and the primers:

-   CBProFpr174: 5′-AGCTGGCTAGCCACTGGGCAGGTAAGTATCA-3′ and-   CBProFpr175: 5′-TGGCGGGATCCTTAAGAGCTGTAATTGAACT-3′

resulting in a 332 bp PCR fragment. The fragment was cut with NheI andBamHI before cloning into pCDNA3.1/HygR (obtained from In Vitro Gen)resulting in PF#34.

The expression cassette for native human blood coagulation factor VIIwas cloned between the BamHI and HindIII sites of PF#34, resulting inplasmid PF#226.

Example 3

Construction of Expression Cassettes Encoding Variant Forms of HumanBlood Coagulation Factor VII that are Having an Additional In VivoGlycosylation Site.

Sequence overhang extension (SOE) PCR was used for generating constructshaving variant human blood coagulation factor open reading frames withsubstituted codons. In the SOE-PCR both the N-terminal part and theC-terminal part of the FVII open reading frame was first amplified inindividual primary PCRs.

For example, in order to change the codons for R315 and V317 to thecodons for N315 and T317 the following primers were used pair vice forthe primary PCR's:

CBProFpr216: 5′-CTTAAGGATCCCGCCACCATGGTCAGCCAG-3′ and CBProFpr229:5′-GGAGTCCCCGGTTTTGTTGGACTGCTGC-3′, and CBProFpr221:5′-ACTTAAGCTTTTATCAAGGGA-3′ and CBProFpr228:5′-GCAGCAGTCCAACAAAACCGGGGACTCC-3′.

The primary PCR products were then combined and the terminal primers(CBProFpr216 and CBProFpr221) added resulting in the secondaryfull-length product encoding the desired R315N+V317T FVII variant. Thesecondary PCR product was trimmed with BamHI and HindIII before cloninginto the vector PF#34 between the BamHI and HindIII sites resulting inPF#249.

Furthermore, in cases where the introduced mutation(s) were sufficientlyclose to a unique restriction endo-nuclease site in the expressionplasmid variant genes were constructed using construction procedureencompassing a single PCR step and a subsequent cloning. For instance,the substitution K143N+N145T was introduced by use of the PCR primers:

-   CBProFpr226: 5′-CATTCTAGAAAACCGGACCGCTAGCAAACC-3′ and-   CBProFpr221: 5′-ACTTAAGCTTTTATCAAGGGA-3′    in a single PCR reaction.

The PCR product was subsequently cloned using the restrictionendo-nuclease sites XbaI and HindIII.

Using the above strategy, the following glycosylation conjugates wereprepared and their amidolytic activities were tested as described in thesection entitled Method of measuring low levels of catalytic activity.Furthermore, some of the conjugates were subjected to the one-stageclotting assay described in the section entitled Method of measuring theclotting activity. The obtained results are compiled in the followingtable.

Glycosylation Conjugate Amidolytic activity Clotting activity T106N + +K143N + N145T + nd V253N + nd R290N + A292T − nd G291N − nd R315N +V317T + + K143N + N145T + + nd R315N + V317T +: Activity measurable; −:Activity not measurable; nd: not determined

Example 4

Improvement of Glycosylation Site Utility by Introduction of AnotherProximal (N-Terminally Located) Glycosylation Site.

In order to prevent autolysis of wild type human FVII a glycosylationsite was introduced at position 315 by making the substitutions: R315Nand V317T as described above, resulting in PF#249.

Upon transfection of CHO K1 cells, using Lipofactamine 2000, lowtransient expression levels were obtained. Assaying the twenty-four hourtransient supernatant by the amidiolytic assay, COASET® FVII, indicatedthat the variant was active. After selection using 400 μg/ml HygromycinB a pool of stable clones was obtained. This pool expresses theR315N+V317T variant at approximately 0.2 μg/ml allowing for Westernblotting analysis for determination of the degree of usage of theintroduced glycosylation site. A twenty-four hour supernatant from thestable pool was assayed by Western blotting and revealed partial usageof the introduced glycosylation site at position 315, approximatelyone-half of the fully processed secreted protein is glycosylated.However, if the native glycosylation site at position 145 is moved toposition 143, by making the substitutions K143N+N145T, the introducedglycosylation site at position 315 is completely glycosylated as judgedby Western blotting.

Example 5

Expression of FVII in HEK 293 Cells

The cell line HEK 293 (ATCC #CRL-1573) was seeded at 20% confluence inT-25 flasks using DMEM, high glucose 10% heat inactivated FCS (Gibco/BRLCat #10091), 5 μg/ml phylloquinone and allowed to grow until confluent.The confluent mono cell layer was transfected with 1, 2, 5, 10 and 20 μgof the plasmid p226 described above using the Lipofectamine 2000transfection agent (Life technologies) according to the manufacturesinstructions. After 24 hours post transfection a medium sample wasdrawn. The FVII concentration in the 24 hour transient expressionexperiments was on average 0.15 μg/ml.

Subsequently, selection medium containing 100 μg/ml Hygromycin B wasadministered to the cells. After three weeks of selection, with renewalof the medium every 3rd or 4th day, the Hygromycin resistant cells wereconfluent in the flasks transfected with 1 μg of plasmid DNA and 2 μg ofplasmid DNA. The cells from each of the five flasks were harvested andpooled. The resulting stable pool of transfectants expressing nativehuman blood coagulation factor VII was frozen in liquid nitrogenaccording to standard procedures.

Example 6

Generation of HEK293 Cells Stably Expressing FVII.

A vial of HEK293 PF#226 transfectant pool was thawed and the cellsseeded in a 75 cm² tissue flask containing 15 ml of DMEM high glucose,10% FCS, phylloquinone (5 μg/ml), 100 U/I penicillin, 100 μg/lstreptomycin, which was used for all subsequent experiments, and grownfor 24 hours. The cells were harvested, diluted and plated in 96 wellmicro titer plates at a cell density of ½ cell/well. After 12 dayscolonies of about 20-100 cells were present in the wells and those wellscontaining only one colony were labeled. After a further two days ofgrowth an additional 100 μl medium was added to all wells. Two dayslater the media in all wells containing only one colony were changed.The first colonies were transferred to 25 cm² tissue flasks culturingafter 3 days and depending on the level of confluency the colonies weretransferred to 25 cm² tissue flasks culturing the next 11 days. Whenconfluent in T-25 tissue flasks the medium was changed and clonesallowed to secrete FVII into the growth medium for 24 hours, after whichthe supernatants were harvested and assayed for the presence of factorVII using the COASET FVII amidolytic assay. One clone, C18, was found toexpress 29 μg/ml FVII.

Example 7

Expression of FVII Glycosylation Variants with No Amidolytic ActivityCapable of Inhibiting the Function of FVIIa

The expression plasmids for expression of the active site ridge mutantsR290N+A292T and G291N were constructed essentially as described inexample 3.

Using the amidiolytic assay COASET® FVII (see above) the ability of thetwo FVII glycosylation variants, R290N+A292T and G291N, to inhibit theactivity of rFVIIa was evaluated. The plasmids PF#250 encodingR290N+A292T and the plasmid PF#294 encoding G291N were transfected intonear confluent serum grown HEK293 cells using Lipofectamin 2000. Thetransfected cells were incubated at 37° C. with 5% CO₂ for three hourspost transfection before the medium was changed to serum free medium(DMEM, ITS-A, ExCyte, Phylloquinone, P/S). Forty hours post transfectionthe conditioned medium was harvested and cleared by centrifugation foranalysis.

A standard curve was made from rFVIIa: 0.0125 ng, 0.025 ng, 0.05 ng,0.075 ng and 0.1 ng. Fifty μl aliquots of undiluted, 2-fold diluted, 5fold diluted, 10-fold diluted and 50-fold diluted condition medium fromeither of the two inactive glycosylation variants R290N+A292T and G291Nor conditioned medium from a mock transfection were spiked with 0.025 ngrFVIIa. When assayed in the COASET FVII assay 0.025 ng of FVIIacorresponded to a signal of OD₄₀₅=0.35 (first run) and OD₄₀₅=0.26(second run).

The obtained results are compiled in the following table:

Glycosylation Conjugate Dilution OD₄₀₅ Standard 0.35 0.26 Mock 50 0.380.19 25 0.31 0.16 10 0.21 0.15 5 0.22 0.14 1 0.08 0.07 R290N + A292T 500.23 — 25 0.12 — 10 0.07 — 5 0.04 — 1 0.04 — G291N 50 — 0.16 25 — 0.0810 — 0.06 5 — 0.05 1 — 0.04

As it appears from the above data the glycosylation conjugates G291N andR290N+A292T inhibit the function rFVIIa.

Example 8

Purification of FVII and Subsequent Activation

Purification of wild-type Factor VII and conjugates thereof wasperformed at 4° C. The supernatant from cells expressing the conjugate(or wild-type FVII) was sterile filtered (0.22 μm) and diluted 2 fold incold milliQ water. EDTA was added to 5 mM, pH adjusted to 8.6 andconductivity was below 10 mS/cm. The sample was applied onto aQ-sepharose Fast Flow resin (Pharmacia) equilibrated at 4° C. in 10 mMTris pH 8.6. After application of the sample the column was washed in 10mM Tris (pH 8.6), 150 mM NaCl, until the absorption at 280 nm reachedbaseline levels. Then the column was equilibrated in 10 mM Tris (pH8.6), 100 mM NaCl. Bound conjugate (or wild-type FVII) was eluted with10 mM Tris (pH 8.6), 100 mM NaCl, 5 mM CaCl₂. Fractions enriched inconjugate (or wild-type FVII) was pooled and concentrated by dialysis orby using Vivaspin concentration units (Vivascience).

Auto-activation of the conjugate (or wild-type FVII) was obtained byconcentration and incubation of the eluted protein in 10 mM Tris (pH7.8-8.6), 100 mM NaCl, 5 mM CaCl₂.

Alternatively, the conjugate (or wild-type FVII) was activated at 37° C.by Factor Xa coupled to CNBr-activated sepharose in 10 mM Tris (pH7.4-8.0) 100 mM NaCl, 5 mM CaCl_(2.)

The conjugate (or wild-type FVIIa) was buffer-exchanged into a solutioncontaining 10 mM CaCl₂, 50 mM NaCl, 3% mannitol, 0.05% Tween80, bufferedat pH 5.6 and sterilfiltered before storage at −80° C.

Example 9

N-Terminal Pegylation of FVII

Factor VII was conjugated with methoxy polyethylene glycol (mPEG) havinga molecular weight of about 5 kDa using M-PEG-CHO (M-ALD-5000, obtainedfrom Shearwater) in a buffer containing 10 mM sodium citrate, 20 mMCaCl₂, 100 mM NaCl, pH 5.5. M-PEG-CHO was present in 50-100-fold molarexcess, and the protein concentration was 0.2-0.5 mg/ml. The reactionwas carried out in 300-1500 μl batches at room temperature for 1 hourwith agitation, and NaBH₃CN was added to 500-1000 fold excess andincubation continued over night with agitation at room temperature.

PEGylated FVII was buffer exchanged to buffer A (10 mM Tris pH 7.6) andapplied at 4° C. on a mono Q column (Pharmacia) equilibrated in bufferA. Bound protein was eluted in a gradient from 0-100% B (10 mM Tris (pH7.6), 500 mM NaCl) over 40 column volumes.

Example 10

Pharmakokinetic Studies in Rats

Both wild-type FVII and the conjugates of the invention are formulatedin 1.3 mg/ml glycyl-glycin buffer pH 5.5 containing 1.5 mg/ml CaCl₂, 30mg/ml mannitol, 0.1 mg/ml polysorbat 80 and 3 mg/ml NaCl. Fordetermination of in vivo half-life each of the preparations areadministered to Sprague-Dawley rats as one intra venous bolus injection.The injections are given slowly over about 10 seconds to reducepotential risk of heart failure due to high Ca²⁺ concentration. Bloodsamples are drawn from each of the nine anaesthetized rats at suitableintervals, e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes and 1 hourafter injection. The blood samples are collected in 1 ml tubescontaining 50 μl Citrate-phosphate-dextrose solution with adenine (Sigma#C4431) in order to prevent coagulation. Immediately after collectionthe samples are stored at about 0° C. until centrifugation andsubsequent collection of the citrate plasma supernatants for assay.Samples are assayed by the one-stage clotting assay as described in thesection Method of measuring the clotting activity and the half-lives arethen calculated.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques, methods, compositions,apparatus and systems described above can be used in variouscombinations. All publications, patents, patent applications, or otherdocuments cited in this application are incorporated by reference intheir entirety for all purposes to the same extent as if each individualpublication, patent, patent application, or other document wereindividually indicated to be incorporated by reference for all purposes.

1. A nucleic acid comprising a nucleotide sequence which encodes apolypeptide comprising an amino acid sequence which (a) differs from thehFVII or hFVIIa sequence SEQ ID NO:1 in 1-15 amino acid residues and (b)comprises an introduced in vivo N-glycosylation site relative to SEQ IDNO:1, wherein the introduced in vivo N-glycosylation site comprises thesubstitution T106N.
 2. The nucleic acid of claim 1, wherein the encodedpolypeptide further comprises at least one additional introduced in vivoN-glycosylation site.
 3. The nucleic acid of claim 2, wherein the atleast one additional introduced in vivo N-glycosylation site comprisesthe substitution I205S/T.
 4. The nucleic acid of claim 2, wherein the atleast one additional introduced in vivo N-glycosylation site comprisesthe substitution V253N.
 5. The nucleic acid of claim 2, wherein the atleast one additional introduced in vivo N-glycosylation site comprisesthe substitutions R315N+V317S/T.
 6. The nucleic acid of claim 2, whereinthe encoded polypeptide comprises two additional introduced in vivoN-glycosylation sites comprising the substitutions I205S/T and V253N. 7.The nucleic acid of claim 2, wherein the encoded polypeptide comprisestwo additional introduced in vivo N-glycosylation sites comprising thesubstitutions I205S/T and T267N.
 8. The nucleic acid of claim 2, whereinthe encoded polypeptide comprises two additional introduced in vivoN-glycosylation sites comprising the substitutions V253N and T267N. 9.The nucleic acid of claim 2, wherein the additional introduced in vivoN-glycosylation site is introduced in a position relative to SEQ ID NO:1occupied by an amino acid residue with more than 25% of its side chainexposed to the solvent.
 10. The nucleic acid of claim 2, wherein theadditional introduced in vivo N-glycosylation site comprises an aminoacid substitution in a position selected from the group consisting of28-48, 139-147, 286-294, 311-319, 338-345 and 388-406 relative to SEQ IDNO:1.
 11. An expression vector comprising the nucleic acid of claim 1.12. A host cell comprising the nucleic acid of claim
 1. 13. The hostcell of claim 12, wherein the host cell is a eukaryotic host cell.
 14. Amethod of producing a conjugate, comprising providing a culturecomprising a eukaryotic host cell capable of in vivo N-glycosylation,said host cell comprising a nucleic acid comprising a nucleotidesequence which encodes a polypeptide comprising an amino acid sequencewhich (a) differs from the hFVII or hFVIIa sequence SEQ ID NO:1 in 1-15amino acid residues and (b) comprises an introduced in vivoN-glycosylation site relative to SEQ ID NO:1, wherein the introduced invivo N-glycosylation site comprises the substitution T106N, and whereinthe nucleic acid is operably linked to a control sequence necessary forexpression of the polypeptide in the host cell; and culturing theculture under conditions which permit expression and N-glycosylation ofthe polypeptide.